Hereditary Spherocytosis and Other Red Cell Membrane Disorders

Hereditary spherocytosis (ANK1, SPTB, SLC4A1, SPTA1 and EPB42 genes), with subsections on hereditary elliptocytosis and pyropoikilocytosis, Southeast Asian ovalocytosis and hereditary stomatocytoses (PIEZO1-related xerocytosis, KCNN4-related Gardos channelopathy, RHAG-related overhydrated stomatocytosis and cryohydrocytosis). The general approach to hereditary haemolytic anaemias, the enzymopathies and SLC4A1-related distal renal tubular acidosis appear only as context and differential diagnosis.

Literature and regulatory information updated: 10 October 2026.

0. Initial Reference Table

1. Introduction

1.1. Concept and Definition of the Entities

Hereditary red cell membrane disorders are constitutional non-immune haemolytic anaemias in which a defect in a membrane skeletal protein, an anchoring protein or an ion transporter shortens red cell survival. They share the underlying clinical features of chronic haemolysis, with anaemia, jaundice, splenomegaly and gallstones, but differ in mechanism, the test that identifies them and, above all, response to splenectomy, which corrects anaemia in spherocytosis and is contraindicated in stomatocytoses [11, 24, 25]. Hereditary spherocytosis (HS) is the most frequent: it results from weakened vertical attachments between the spectrin skeleton and lipid bilayer, with membrane loss in microvesicles, a reduced surface area-to-volume ratio and formation of poorly deformable spherocytes that are retained and destroyed by the spleen [5, 6]. The term spherocyte denotes a spherical red cell without central pallor, with a reduced surface area-to-volume ratio [2].

Hereditary elliptocytosis (HE) results from defects in horizontal skeletal interactions, particularly self-association of spectrin dimers into tetramers and the spectrin–actin–protein 4.1R junctional complex; the red cell loses its ability to regain a disc shape and becomes elliptical [3, 5]. Hereditary pyropoikilocytosis (HPP) is the severe biallelic form of HE, with fragmentation, microspherocytosis and micropoikilocytosis from the neonatal period [5]. Southeast Asian ovalocytosis (SAO) is caused by a heterozygous nine-amino-acid deletion in band 3 that makes the membrane rigid; it is generally asymptomatic and confers protection against malaria [5, 26]. Hereditary stomatocytoses (HSt) are disorders of cation permeability: a primary Na+ and K+ leak exceeding the capacity of the Na+/K+-ATPase pump alters cell hydration and volume. They comprise dehydrated stomatocytosis (DHSt) or hereditary xerocytosis, overhydrated stomatocytosis (OHSt), cryohydrocytosis (CHC) and familial pseudohyperkalaemia (FP) [5, 27].

1.2. Historical Milestones and Molecular Discovery

The first description of microspherocytes in a blood film from an anaemic patient dates to 1871, according to the review by de Wilde and colleagues [7]. The genetic family study published by Morton and colleagues in 1962 established disease transmission and provided the US birth prevalence estimate still recorded by Orphanet, 20 per 100,000 [1, 28]. Before 1990 it was known that red cells in dominant or recessive HS were deficient in spectrin to a degree correlated with severity, and that an HS locus existed at 8p11. In 1990, Lux and colleagues mapped the erythroid ankyrin gene (ANK1) to 8p11.2 and demonstrated loss of one copy in two unrelated children with severe HS and heterozygous del(8)(p11-p21.1) deletions, with ankyrin deficiency; this was the first association of HS with a gene [29]. In 1996, Eber and colleagues screened ANK1 (42 exons and promoter) and SLC4A1 (19 exons) in 46 families, identified 12 ANK1 and 5 SLC4A1 variants, and estimated that ANK1 accounted for 35–65% and band 3 for 15–25% of HS in Northern Europe; null variants predominated in the dominant form and ANK1 missense and promoter variants in the recessive form [30].

The molecular biology of other membrane disorders followed a parallel course. In 1991, Jarolim and colleagues identified deletion of band 3 codons 400–408 in all 30 individuals with ovalocytosis studied, from Malaysia, the Philippines and two coastal regions of Papua New Guinea, and in none of 50 controls [26]. In 1995, Maréchal and colleagues described the ethnic distribution of the low-expression α-spectrin allele αLELY, with allele frequencies of 31% in Caucasians, 21% in Africans, 20% in Japanese and 22% in Chinese populations [31]. In 2005, Bruce and colleagues showed that single substitutions in the band 3 transport domain convert the anion exchanger into an unregulated cation channel, with a marked Na+ and K+ leak at 0 °C, in 11 families with dominant stomatocytosis or spherocytosis [32]; in 2009, the same group attributed OHSt to heterozygous RHAG variants that open a cation pore in the Rh-associated glycoprotein [33]. In 2012, Zarychanski and colleagues linked hereditary xerocytosis to PIEZO1 variants, the first human disease associated with a mammalian mechanotransduction channel [34]. In 2013, Albuisson and colleagues demonstrated that PIEZO1 variants cause gain of function, with slower channel inactivation [35], and Andolfo and colleagues described 11 variants in 7 families and the first association between PIEZO1 and perinatal oedema [36]. In 2015, Rapetti-Mauss and colleagues identified the first human Gardos channel disorder (KCNN4) [37], confirmed in the same year by Andolfo and colleagues in a second series [38]. Osmotic gradient ektacytometry, developed in the 1980s by Bessis, Mohandas and Feo, was reimplemented in commercial instruments and is now the reference functional test at several European centres [7, 39].

1.3. Nomenclature and Reclassifications

OMIM numbers HS by gene: type 1 (ANK1), type 2 (SPTB), type 3 (SPTA1), type 4 (SLC4A1) and type 5 (EPB42) [2, 3]. Elliptocytoses follow the same logic (type 1 EPB41, type 2 SPTA1, type 3 SPTB), and the historical designation “Rhesus-linked elliptocytosis” derives from the proximity of the EPB41 locus to the RH locus on 1p [3]. Hereditary xerocytosis is synonymous with dehydrated hereditary stomatocytosis; the French group proposed “Gardos channelopathy” for the KCNN4 form because these patients frequently lack clear laboratory signs of dehydration [18]. Many variants retain historical protein or city names (protein 4.2 Nippon, Komatsu, Nancy, Lisboa; ankyrin Florianópolis; band 3 Courcouronnes, Campinas, Genas; spectrin αLEPRA, from Low Expression PRAgue, and αLELY, from Low Expression LYon), which should be retained alongside Human Genome Variation Society (HGVS) nomenclature to facilitate reading of the literature [2, 5, 6].

1.4. Scope of the Article

This article treats HS as the main entity and devotes separate subsections to HE and HPP, SAO and the stomatocytoses (PIEZO1, KCNN4, RHAG, cryohydrocytosis and Rh deficiency syndrome), because these entities share screening tests with HS and because confusion between them has therapeutic consequences. The general approach to hereditary haemolytic anaemias, the enzymopathies, the congenital dyserythropoietic anaemias (CDA, congenital dyserythropoietic anemia) and SLC4A1-related distal renal tubular acidosis (dRTA) appear here only as context, allelic disorder and differential diagnosis [23].

2. Molecular Genetics and Pathophysiology

2.1. Architecture of the Red Cell Membrane

The red cell membrane consists of an asymmetric lipid bilayer of cholesterol and phospholipids traversed by integral proteins, and a spectrin skeleton covering approximately 65% of the cytoplasmic surface without direct contact with lipids [5, 6]. Spectrin is a heterodimer of α (SPTA1) and β (SPTB) chains; dimers associate head-to-head into tetramers, and tetramer ends bind to short actin filaments through protein 4.1R (EPB41), forming the junctional complex. These are the horizontal interactions, which confer mechanical strength on the skeleton. Vertical interactions attach the skeleton to the bilayer through two complexes: the band 3–ankyrin–protein 4.2 complex, in which ankyrin-1 (ANK1) links β-spectrin to band 3 (SLC4A1) with stabilisation by protein 4.2 (EPB42), and the glycophorin C–p55–protein 4.1R complex [2, 5]. Band 3 also forms part of a macrocomplex with Rh-associated glycoprotein (RhAG), Rh polypeptides and CD47 [2, 3]. High intracellular K+ and low Na+ are maintained by the Na+/K+-ATPase pump; stomatocytosis develops when passive cation leakage exceeds pump capacity [5].

2.2. Pathophysiology of Spherocytosis

In HS, deficiency or dysfunction of band 3, protein 4.2, ankyrin, α-spectrin or β-spectrin weakens vertical interactions. Local uncoupling between skeleton and bilayer releases lipid microvesicles; the red cell loses surface area without losing volume, becomes spherical, dehydrated and poorly deformable, and is retained in the splenic cords, where it is phagocytosed [5, 6]. Two biochemical patterns predominate: ankyrin and spectrin deficiencies, in which ankyrin loss brings a proportional reduction in spectrin, and band 3 deficiencies, with secondary protein 4.2 deficiency [3, 6]. Severity tracks the degree of deficiency: severe deficiency of one protein, or an imbalance with combined deficiencies, produces more severe haemolysis, and partial spectrin deficiency tends to produce a moderate-to-severe phenotype [6]. HS red cells also have increased cation permeability, with Na+ influx and K+ loss contributing to dehydration and elevated MCHC; modulation by PIEZO1 and the Gardos channel has been proposed to explain the HS1 (dehydrated) and HS2 (partially hydrated) ektacytometry profiles [39, 40].

2.3. Spherocytosis Genes, Gene by Gene

Source: GeneReviews, Table 3a [2].

ANK1 (8p11.21) is the most frequent cause of HS, responsible for about half of cases according to NCBI Gene, and encodes the core of the ankyrin-1 complex, which links the spectrin–actin skeleton to band 3, protein 4.2 and Na+/K+-ATPase [3]. The muscle isoforms link the sarcoplasmic reticulum to the myofibrils, but no muscle phenotype is recorded in OMIM, ClinGen or GenCC [3]. ClinGen classifies the ANK1–HS relationship as “definitive” for AD inheritance (24 March 2021) and “limited” for AR inheritance (23 June 2021) [3]. In the 2006 review summarised by the British guideline, ANK1 variants were mainly frameshift (17) and nonsense (8), with aberrant splicing (4), missense (4) and promoter (2); loss of one haploid set gives a combined deficiency of spectrin and protein 4.2, and the combination of a promoter variant in trans with a coding variant gives recessive HS [6]. De novo variants are found mainly in apparently recessive HS associated with ANK1 and SPTB and are then transmitted dominantly; ankyrin Florianópolis, a recurrent frameshift variant, was found in three unrelated probands of different genetic origins with severe dominant HS [6]. In 2026, the French AURAGEN consortium described deep intronic ANK1 variants causing pseudoexon inclusion, identified by genome sequencing and confirmed by functional study; these are variants that exon-centred panels and exome sequencing do not detect [41].

SPTB (14q23.3) is associated with two overlapping phenotypes: autosomal dominant HS type 2, due to null variants silencing one allele, and elliptocytosis type 3 with fatal or near-fatal neonatal haemolytic anaemia, with AD or AR inheritance [3, 6]. In the 2006 review, SPTB variants in HS were null (10), nonsense or non-coding (10), missense (5) and one polymorphism [6]. In a Utrecht series of 233 patients studied by ektacytometry, median maximal elongation index (EImax) was lower in the SPTB group (0.509) than in the SLC4A1 (0.557) and SPTA1 groups (0.564; p<0.01), and non-missense variants were associated with lower deformability; these are group medians and cannot identify the gene in an individual [7].

SLC4A1 (17q21.31) encodes band 3, the Cl−/HCO3− exchanger of the red cell membrane and, as a renal isoform, of the basolateral membrane of the α-intercalated cell in the collecting duct; in red cells it transports CO2 as bicarbonate and anchors the skeleton through ankyrin [3, 23]. In autosomal dominant HS type 4, variants are missense (23), nonsense or frameshift (18) and other types; the mechanism is mutant mRNA instability or failure to incorporate the protein into the membrane, with band 3 deficiency and, in some patients, partial reduction of band 6 [6]. There is no founder effect, but co-inheritance of two unequally expressed alleles has an additive effect and worsens the condition [6]. “Mushroom” cells on the blood film are associated with band 3 defects [5]. Transport domain variants (exon 17) cause a particular form, spherocytosis with low-temperature leak (SphLTL), with haemolysis disproportionate to the small reduction in band 3 and, in some patients, pseudohyperkalaemia, because mutant band 3 functions as a cation transporter [6, 32]. SLC4A1 variants also define antigens of the Diego and Wright, Waldner, Swann and Froese systems, which are not diseases [3].

SPTA1 (1q23.1) causes HS type 3, which is always recessive. Because α-spectrin is synthesised in excess relative to β-spectrin, only a substantial reduction in both alleles produces clinical deficiency; the phenotype follows the total quantity of residual functional α-spectrin [3, 42]. In the series of 11 patients by Chonat and colleagues, the spectrum ranged from moderately severe HS to transfusion dependence and fetal hydrops, fatal if transfusion was not started before term; patients with the αLEPRA allele in trans with a null allele were not always transfusion-dependent and improved or became free of anaemia after partial or total splenectomy, whereas patients with near-complete α-spectrin deficiency survived only with early or intrauterine transfusion and remained transfusion-dependent after splenectomy [3, 42]. Qualitative defects in the αI self-association domain, by contrast, produce elliptical and poikilocytic phenotypes [3, 5].

EPB42 (15q15.2) encodes protein 4.2, which binds ATP, stabilises the band 3–ankyrin interaction and binds to the C-terminal EF-hands of α-spectrin in a calcium- and calmodulin-dependent manner; the disease mechanism is loss of function, with AR inheritance [2, 3]. Heterozygotes are asymptomatic [2]. Absence of membrane protein 4.2 may also be secondary to biallelic SLC4A1 variants that reduce band 3 or affect its protein 4.2 binding site [2, 5]. EPB42-related HS is typically milder than recessive ANK1 or SPTA1 forms [2]. Orphanet assigns AD inheritance to HS overall, whereas gene-specific submissions in GenCC (Labcorp/Invitae and PanelApp Australia, both “Strong”) record AR inheritance for EPB42; ClinGen dosage curation classifies the gene as associated with a recessive phenotype [3].

2.4. Low-Expression Alleles, De Novo Variants and Modifiers

Variability of HS within a family is often explained by low-expression alleles co-inherited in trans. The SPTA1 αLEPRA allele, a splicing variant with a frequency of 3.6% in the normal population, is silent in isolation, but in homozygosity or compound heterozygosity with an SPTA1 HS allele produces severe HS with very low spectrin, and is the most prevalent allele in non-dominant HS [5, 6]. The αLELY allele has two changes in cis, a missense change in exon 40 (Leu→Val at residue 1857/1858) and a C>T substitution at position −12 of intron 45, causing partial exon 46 skipping and reducing functional α-spectrin produced by that chromosome to approximately half [3, 31]. It is not a disease allele: it is asymptomatic in isolation because α-spectrin is synthesised in excess, but in trans with an α-elliptocytosis allele it reduces the fraction of normal spectrin and converts mild HE into HPP [5, 31]. In band 3, a low-expression allele (Genas) has been associated with intrafamilial variability [5]. Gilbert syndrome, caused by the UGT1A1 promoter polymorphism (TA)7, increases bilirubin and gallstone risk in children with HS and may create a false impression of severe haemolysis; approximately 50% of Europeans and North Americans are heterozygous and 9% homozygous for this allele [6, 43]. Co-inheritance of haemochromatosis alleles (HFE) increases iron overload risk in adults with chronic haemolytic anaemia [5, 6].

2.5. Elliptocytosis and Pyropoikilocytosis

HE results from quantitative protein 4.1R deficiency or qualitative spectrin variants affecting dimer self-association (αI domain, αI/80 peptide and others) [5]. According to a tertiary review cited by GeneGuidance, SPTA1 accounts for approximately 65% of elliptocytosis, SPTB approximately 30% and EPB41 approximately 5% [3]. Spectrin defects are more frequent in populations of African ancestry and are associated with a more severe phenotype, whereas protein 4.1 deficiencies are more frequent in Caucasians [5]. The spectrin αI/74 variant may result from SPTA1 variants or variants in exon 30 of SPTB [5]. The consanguineous family described with a homozygous 50 kb EPB41 deletion removing both initiation sites had complete absence of protein 4.1R and homozygous elliptocytosis, with asymptomatic heterozygous parents [3]. HPP is biallelic; in most patients, the severe phenotype is explained by the αLELY allele in trans with an elliptocytosis allele, and microspherocytosis reflects reduced α-spectrin [5]. Unlike recessive HS, in most HE and HPP at least one parent has an HE phenotype [5]. In an Indian series of 10 patients with 12 SPTA1 variants, elliptocytosis accompanied monoallelic variants and HPP biallelic variants [3].

2.6. Southeast Asian Ovalocytosis

SAO is caused by the heterozygous 27-bp deletion in SLC4A1 that removes amino acids 400–408 of band 3, at the boundary between the cytoplasmic and the membrane domain; it is linked in cis to the substitution NM_000342.4(SLC4A1):c.166A>G p.(Lys56Glu), the Memphis polymorphism [26]. The mutant band 3 makes the membrane rigid and confers resistance to invasion by several malaria parasites [3, 26]. The homozygous state is almost always lethal in utero: the 2021 French PNDS counts six reported homozygotes, with severe transfusion-dependent anaemia from fetal life and renal tubular acidosis [8]. Isolated SAO does not cause dRTA, but compound heterozygosity of SAO with another SLC4A1 variant is a frequent cause of recessive dRTA with haemolytic anaemia in Southeast Asia [3, 5].

2.7. Hereditary Stomatocytoses

PIEZO1 (16q24.3) encodes a non-selective cation channel activated by membrane tension, a homotrimer that converts mechanical force into Ca2+ influx; it is required for vascular and lymphatic development and red cell volume regulation [3]. In xerocytosis, heterozygous gain-of-function variants delay channel inactivation; Ca2+ influx activates the Gardos channel, with K+ and water loss and cellular dehydration [18, 35]. Variants cluster in exon 51 (pore and C-terminal region), with secondary hotspots in exons 14–18 (extracellular blade) and 42–45 [18]. Hepatic iron overload in xerocytosis is directly linked to the physiological role of PIEZO1 in the liver and macrophages [27]. PIEZO1-mediated dehydration is associated with protection against malaria, and a common PIEZO1 allele exists in populations of African ancestry [3, 18]. Missense variants in the extracellular domain define antigens of the Er blood group system (Era, Erb, Er3, Er4, Er5); anti-Er4 and anti-Er5 have been implicated in severe haemolytic disease of the fetus and newborn [3].

KCNN4 (19q13.31) encodes KCa3.1, the Gardos channel, an intermediate-conductance K+ channel activated by Ca2+ through calmodulin and independent of voltage [3]. Gardos channelopathy variants are heterozygous gain-of-function missense variants (the recurrent p.(Arg352His), which also arises de novo, and p.(Val282Met), among others) or small in-frame or splicing deletions; they increase channel activity and its sensitivity to Ca2+, but the mutant channel remains inhibitable [3, 37, 38]. Some de novo variants (p.(Val222Leu), protein designation published on NP_002241.1; NM_002250.3:c.1018C>A p.(His340Asn)) increase Ca2+ sensitivity but cause anaemia with stomatocytosis without dehydration [3].

RHAG (6p12.3) encodes RhAG (CD241), an obligatory partner of the RhD and RhCE polypeptides in the Rh complex, an ammonium transporter and possible CO2 channel [3]. The heterozygous p.(Ile61Arg) and p.(Phe65Ser) variants in OHSt open a cation pore, with leakage approximately six times that of wild type in Xenopus oocytes, very high intracellular Na+ and low K+ [33]. Biallelic loss of RHAG function causes regulator-type Rh deficiency syndrome, with absence of all Rh antigens [3]. Cryohydrocytosis results from variants in the band 3 C-terminal domain (nine substitutions between amino acids 687 and 796) and, in the more severe form, from SLC2A1 (GLUT1) variants, with marked haemolysis, pseudohyperkalaemia and the paediatric neurological syndrome of GLUT1 deficiency; familial pseudohyperkalaemia has been associated with ABCB6 [5]. ABCB6 was also included, alongside PIEZO1 and KCNN4, in the genetic spectrum of stomatocytoses in the 2025 review [27].

Source: ICSH 2015, Table 5, with the locations published in 2015 [5]; KCNN4 was not included in the table and was associated with DHSt in the same year [37]. Current cytobands are in section 0.

2.8. Variant Spectrum and Nomenclature

HS variants are mostly private to each family; exceptions are founder EPB42 alleles in Japan, SPTB HE variants in Southeast Asia and the SAO deletion [2, 6, 44]. The following table brings together the named variants used in this article, normalised in ClinVar and VariantValidator against GRCh38 and the MANE Select (Matched Annotation from NCBI and EMBL-EBI) transcript [45]. For EPB42, the MANE transcript (NM_001114134.2) corresponds to the short isoform; historical and GeneReviews numbering (NM_000119) correspond to the long isoform, with 30 additional codons, so positions differ by 90 nucleotides and 30 amino acids [2, 45].

ClinVar classifications are those recorded on 10 October 2026; EPB42 and SLC4A1 rows without their own citation key derive from the same ClinVar and VariantValidator consultation [2, 23, 45]. The correspondence of records 1030630 and 258948 to αLEPRA and αLELY was inferred from the ClinVar index rather than a primary publication using HGVS notation and should be interpreted with that qualification. The 4.2 Lisboa allele (a single-nucleotide deletion at nucleotide 264 or 265, codon 88 or 89, exon 2 of EPB42) was not normalised because the publication does not specify the transcript [58]. The SPTA1 c.7134G>A variant in the case in section 10 was described without a transcript; assuming NM_003126.4, it corresponds to p.(Gln2378=), a synonymous substitution at the last exonic position with a predicted effect on splicing [12, 45].

2.9. Genotype–Phenotype Correlations, Penetrance and Expressivity

Correlations are clearer for inheritance than for the gene. Dominant forms due to ANK1, SPTB and SLC4A1 tend to be mild to moderate, and expression is relatively uniform within a family and variable between families [2, 6]. Recessive forms due to SPTA1 and ANK1 and the rare recessive forms of SLC4A1 and SPTB are severe, often transfusion-dependent and sometimes fetal [2, 42]. For EPB42, homozygosity for p.Ala142Thr (4.2 Nippon; historical numbering, = NM_001114134.2:c.334G>A p.(Ala112Thr)) gave moderately severe HS with haemoglobin (Hb) as low as 6.1 g/dL in Japanese patients, and an Italian patient with the same genotype had moderate haemolytic anaemia from birth; homozygosity for p.Ala142Thr or p.Asp175Tyr (4.2 Komatsu; historical numbering, = NM_001114134.2:c.433G>T p.(Asp145Tyr)) can give atypical HS with ovalostomatocytes and few spherocytes, which improves after splenectomy, and compound heterozygosity with another variant gives typical HS [2]. In the Chinese series of 94 patients, SPTB and ANK1 variants were mainly nonsense and frameshift and those in SLC4A1, SPTA1 and EPB42 mainly missense; patients with SLC4A1 had higher MCHC, and no other correlations were found [59]. In a series of 55 Chinese patients, children aged 3 years or younger had lower Hb, red cell count and absolute reticulocytes, with no phenotypic differences between ANK1 and SPTB [60]. In the Milan series of 300 patients classified by protein electrophoresis, spectrin deficiency was the most frequent in those diagnosed in childhood and was associated with slightly lower Hb and more haemolysis, and band 3 deficiency was the most frequent in those diagnosed in adulthood [61]. Factors outside the primary gene (low-expression alleles, UGT1A1, HFE, co-inheritance of thalassaemia trait or glucose-6-phosphate dehydrogenase, G6PD, deficiency) modulate expression [5, 30, 44].

In xerocytosis, haematological penetrance is high but expressivity is highly variable: in the French series, 68% of patients had no anaemia, and the duplication p.Leu2495_Glu2496dup was associated with lower Hb (121.3±11.1 g/L) than NM_001142864.4:c.7367G>A p.(Arg2456His) (130.6±14.5 g/L) and than the other variants (143.7±16.7 g/L; p<0.05), with no clear genotype–phenotype correlation overall [18]. In Gardos channelopathy, expressivity ranges from severe anaemia to absence of anaemia in carrier parents [3].

2.10. Allelic Disorders and Contiguous Gene Syndromes

SLC4A1 is the richest example of allelic disorders: HS type 4 (AD), SAO (AD), cryohydrocytosis (AD), dominant dRTA (179800) due to dominant-negative variants such as NM_000342.4:c.1766G>A p.(Arg589His), c.1765C>T p.(Arg589Cys) and c.1838C>T p.(Ser613Phe), usually with normal red cell morphology, and recessive dRTA with haemolytic anaemia (611590), mainly in Southeast Asia, often with the SAO allele on one chromosome [3, 23]. In a European cohort of 340 patients with primary dRTA, nephrolithiasis was more frequent with SLC4A1 variants (42% versus 21%), and the complete band 3 null state (for example, homozygosity for NM_000342.4:c.1430C>A p.(Ser477Ter)) causes severe transfusion-dependent haemolytic anaemia, dyserythropoiesis and complete dRTA [3]. SLC4A1-related dominant dRTA predominates in temperate zones and the recessive form in tropical regions from Oman to Papua New Guinea [5]. Band 3 Edmonton I has been described as a simultaneous cause of spherocytosis and dRTA [23].

PIEZO1 has two disorders with opposite mechanisms: gain-of-function xerocytosis (AD) and generalised lymphatic dysplasia with non-immune fetal hydrops due to biallelic loss of function (AR; approximately 10 cases), with uniform lymphoedema, intestinal or pulmonary lymphangiectasia, pleural effusions, chylothorax and pericardial effusions [3, 22]. PIEZO1 also accounts for a proportion of Hennekam-type generalised congenital lymphoedema, in which approximately 25% of cases are due to CCBE1 [62]. In the French xerocytosis series, two families showed overlap with lymphoedema [18]. RHAG causes OHSt (AD) and Rh deficiency syndrome (AR) [3]. Among contiguous gene syndromes, an 8p11.2 deletion or translocation including ANK1 produces spherocytosis with dysmorphism or neurological impairment [3, 5], and a contiguous Xq22.3 deletion including COL4A5 and ACSL4 (FACL4) causes Alport syndrome with intellectual disability, midface hypoplasia and elliptocytosis, with X-linked inheritance [3, 63]. EPB42 has no other known phenotype [2].

3. Clinical Spectrum and Manifestations by System

3.1. Severity Spectrum of Spherocytosis

HS ranges from an asymptomatic carrier state, with normal Hb and bilirubin and no spherocytes, to severe transfusion-dependent haemolysis; most patients have mild disease, and 20–30% have purely compensated haemolysis, with normal Hb and reticulocytosis [5, 6]. The British guideline uses a modified Eber classification (1990), and GeneReviews a similar classification based on Eber and Lux (2004). Their thresholds do not coincide and should be interpreted as severity guidance rather than criteria.

Source: BSH 2011, Table II; spectrin quantification (normal 226±54 × 10³ molecules per cell) is informative only [6].

Source: GeneReviews, Table 1 [2]. The 3-year threshold for splenectomy in severe disease predates the 2017 European recommendations, which advise against splenectomy before 5 years (section 7.6) [11].

The 2021 French PNDS (National Diagnosis and Care Protocol) uses a third classification, with the proportion of patients in each class [8].

Source: PNDS 2021, summary, section 1.1 on spherocytosis and appendix 10 [8]. Severity is usually similar within the same family, except when an additional factor, such as co-inherited β-thalassaemia trait or sickle cell trait, modifies the clinical expression [8].

In a Milan cohort of 204 non-splenectomised patients diagnosed between 2014 and 2022, classified as asymptomatic (Hb >14 g/dL in men and >12 g/dL in women), mild (>11 g/dL), moderate (8–11 g/dL) or severe (<8 g/dL), the HS2 ektacytometry profile (right-shifted curve) was associated with younger patients (median 10 versus 22 years; p=0.002), more severe forms (13.7% versus 2.6%) and fewer mild forms (17.6% versus 36.6%) than the HS1 profile [40].

3.2. Fetal and Neonatal Period

Severe recessive SPTA1, ANK1 or SLC4A1 forms may present with fetal anaemia and non-immune hydrops; fatal hydrops is rare and more likely with a family history [5, 42]. In an American series of four at-risk pregnancies published in 2026, a fetus homozygous for an SPTA1 variant had severe anaemia and hydrops and died in utero; the child of a mother heterozygous for an ANK1 variant had fetal anaemia and polyhydramnios and required four transfusions after birth; the child of a mother heterozygous for SPTB had unremarkable fetal surveillance but required quintuple phototherapy; and the fourth, also born to a mother with SPTB and with a family history of neonatal bilirubin toxicity, had no complications [64].

Neonatal jaundice is the most common presentation. It usually appears in the first days, sometimes in the first 24 hours, may require phototherapy or exchange transfusion and bears no clear relation to later severity [2, 5, 6]. The bilirubin peak may occur after several days, so the newborn of a parent with HS should be monitored for several days [6]. In a study cited by the British guideline, 4 of 402 jaundiced newborns had HS, about 1% of this selected group, compared with 1/2000 to 1/5000 in the general population [6]. HS is the third haemolytic cause of neonatal jaundice, after ABO isoimmunisation and G6PD deficiency, and the most common cause of non-immune haemolytic hyperbilirubinaemia in newborns with kernicterus, according to a 2025 review [65]. According to the French PNDS (National Diagnosis and Care Protocol), neonatal jaundice occurs in 60–90% of newborns with HS, depending on the series, half before 48 hours of life, and HS accounts for 3–7% of the causes identified in cohorts with severe hyperbilirubinaemia at risk of kernicterus or with an indication for exchange transfusion; co-inheritance of Gilbert syndrome increases the risk [8]. The protocol calls for investigation of haemolysis in the presence of early hyperbilirubinaemia (within the first 24 hours), pathological hyperbilirubinaemia (TSB at or above the 95th percentile for age) or hyperbilirubinaemia prolonged beyond the first week, with ABO incompatibility, G6PD deficiency and PK deficiency as the main differentials [8]. Hb is usually normal at birth and falls rapidly in the first month; anaemia is most severe in the first 4–6 months, owing to an inadequate erythropoietic response, and may require repeated transfusions that do not predict later severity [2, 5, 6]. In the PNDS, the strong initial reticulocytosis falls in the first two weeks, about 40% of newborns with HS need a transfusion in the first month and, depending on the series, half to two thirds are transfused before 2 months; transfusion may be needed in the first 6–8 weeks without predicting later severity, and hydrops fetalis is exceptional [8]. Transfusion dependence beyond the first year is unusual, and many children with Hb of 50–60 g/L do not need transfusion [6]. Neonatal cholestatic jaundice due to excessive haemolysis is rare and more likely in severe hereditary haemolytic anaemias [2].

In elliptocytosis, transient infantile poikilocytosis causes transfusion-dependent anaemia up to the first year, which evolves rapidly to the mild phenotype of the parent [5]. The PNDS describes this transient pyropoikilocytosis of the newborn and infant, in which usually one parent has simple HE and the other a normal blood film: there is haemolytic jaundice at birth, about 40% need a transfusion in the first month, and haemolysis persists over the first 12–24 months, with moderate anaemia (8–11 g/dL) and marked reticulocytosis, before evolving to simple HE [8]. In a Thai population with founder SPTB variants, heterozygous HE was an independent risk factor for jaundice requiring phototherapy, with a relative risk (RR) of 5.01 (95% confidence interval, 95% CI, 2.56–9.79), and bilirubin in the first three days was higher than in newborns with G6PD deficiency [44]. In xerocytosis, neonatal anaemia is more marked than later, and perinatal oedema is a distinctive manifestation (section 3.9) [18].

3.3. Childhood and Adolescence

The classical triad is anaemia, jaundice and splenomegaly [3]. Anaemia reflects the balance between haemolysis and spleen size; many patients have compensated haemolysis [6]. Jaundice is intermittent and worsens with infections; hyperbilirubinaemia is unconjugated and only rarely conjugated, due to biliary obstruction [2]. Mild-to-moderate splenomegaly is present in most patients and apparent in early childhood, but spleen size alone is not an indication for splenectomy; splenic rupture is no more frequent than in the general population and there is no evidence supporting restriction of physical activity in children with HS-related splenomegaly, although GeneReviews advises against contact sports when splenomegaly is present and considers acute or excessive splenomegaly a greater risk than mild chronic splenomegaly [2, 6]. Mild HS may worsen in disorders associated with splenomegaly, such as infectious mononucleosis [6]. Young children may have age-related iron deficiency, but anaemia does not fully resolve with iron and reticulocytosis persists [2]. In an Israeli cohort of 123 children followed between 1995 and 2025, median age at diagnosis was 0.2 years, 65% were diagnosed in the first year and 84.6% before 4 years; 81.3% had a family history, mean baseline Hb was 9.8±1.4 g/dL, reticulocytes 7.9±2.9%, 91.9% had splenomegaly, 29.3% underwent splenectomy and 41.5% cholecystectomy [9]. Approximately 75% of patients have a family history of HS [6].

Transient aplastic crisis is the most characteristic acute complication. It is caused mainly by parvovirus B19, which interrupts erythropoiesis for 10–14 days; in a population with accelerated haemolysis, this arrest produces a rapid fall in Hb with very low reticulocytes [5, 66]. In the Israeli cohort, 61 of 123 children (49.6%) had an aplastic crisis, at a median age of 6.6 years (interquartile range 4.6–8.2); in 3 it was the initial presentation, and none occurred after splenectomy [9]. Acute parvovirus B19 was confirmed in 94.4% of those tested. At the nadir, mean Hb was 5.6±1.0 g/dL and reticulocytes 0.4±0.2%; 63.6% had leucopenia, 61.8% neutropenia and 50.9% pancytopenia. Pancytopenia was associated with lower Hb (5.3 versus 5.9 g/dL; p=0.009) and more transfusions (median 2 versus 1 unit; p<0.001). No baseline factor predicted the occurrence of the crisis, but each additional percentage point of baseline reticulocytes increased the probability of pancytopenia (adjusted odds ratio, OR, 1.63; 95% CI 1.08–2.46) and of more than one transfusion (OR 1.75; 95% CI 1.18–2.60), and each year of age increased the probability of more than one transfusion (OR 1.45; 95% CI 1.09–1.92). All children recovered, without sustained marrow failure [9]. In a Czech multicentre study conducted during the 2023–2024 European parvovirus B19 epidemic, 23 non-splenectomised children with HS, with a mean age of 6.9 years, were admitted for aplastic crisis requiring transfusion; mean Hb was 4.93 g/dL (2.9–7.4), 26% had pancytopenia, anti-B19 IgM was positive in all, and IgM cross-reactivity with cytomegalovirus (CMV) or Epstein-Barr virus (EBV) occurred in 47.8%, which required confirmation by polymerase chain reaction (PCR). Sixty-three units were transfused (median 2 per child), and in a 6-year-old child the crisis revealed HS [66]. That study defined aplastic crisis as a fall in Hb ≥3 g/dL with reticulocytes <25 × 10⁹/L [66]. The French PNDS (National Diagnosis and Care Protocol) describes direct infection of erythroblasts by parvovirus B19 for about 3 weeks (the ICSH refers to 10–14 days of erythropoietic arrest), with reticulocytes usually below 50 × 10⁹/L, one or several transfusions often needed, lifelong immunity after primary infection and simultaneous cases in several family members; when the crisis reveals the disease, the haemolysis work-up is performed more than 2 months after the episode and the transfusion [5, 8]. Infections with CMV, human herpesvirus 6 or EBV can also acutely increase haemolysis [5].

3.4. Gallstones

Pigment gallstones occur in 21–63% of patients with HS, and many children develop them in the first decade; in an Italian survey of 468 children with HS, 79 had gallstones and approximately half were diagnosed before 11 years [6]. Co-inherited Gilbert syndrome increases gallstone risk fivefold [6]. Complications are biliary colic, cholecystitis, obstructive jaundice and pancreatitis [2]. After splenectomy, patients stop forming pigment stones: 17 patients splenectomised before 18 years, without stones at surgery, did not develop gallstones over a mean follow-up of 15 years [6]. Recurrent abdominal pain with haemolytic anaemia enters the metabolic differential diagnosis alongside sickle cell disease, acute porphyrias and paroxysmal nocturnal haemoglobinuria [67].

In the French PNDS (National Diagnosis and Care Protocol), biliary or gallbladder lithiasis affects one third of children aged 5 to 13 years and 40–50% of adults aged 20 to 50 years, classically appears between 5 and 15 years and is often asymptomatic, discovered on a routine ultrasound; Gilbert syndrome, with a prevalence of 11–19% in European and African populations, multiplies the risk by 4 to 5 and gives marked unconjugated hyperbilirubinaemia, above 50 or even 100 µmol/L [8]. Pain in the right upper quadrant or epigastrium, especially postprandial and after rich meals, is the usual symptom, and cholecystitis and cholangitis are reasons for urgent admission [8].

3.5. Adults, Older People and Iron Overload

HS may be diagnosed at any age, including the seventh–ninth decades, often after an infection [5, 6]. An adult whose condition was not recognised in childhood presents with mild (Hb 11–15 g/dL) or moderate (8–11.5 g/dL) chronic haemolytic anaemia, with jaundice, splenomegaly and gallstones at a relatively young age [2]. In the Milan series, band 3 deficiency was the most frequent defect in patients diagnosed as adults [61]. Adults with HS have above-normal erythropoietin levels [6]. Chronic anaemia increases intestinal iron absorption and, with coinheritance of haemochromatosis alleles, severe overload may occur even without transfusions [6]. Hyperferritinaemia is much more frequent in xerocytosis than in HS [18].

The French PNDS (National Diagnosis and Care Protocol) adds rare complications of chronic haemolysis, mainly in non-splenectomised patients: skin ulcers and foci of extramedullary haematopoiesis, which may reveal the disease; extramedullary haematopoiesis, exceptional in HS, is often treated with splenectomy and sometimes with regular transfusions combined with hydroxycarbamide [8]. Splenic infarction is rare and should prompt a search for an associated haemoglobinopathy (for example, sickle cell trait), thrombophilia or G6PD or PK deficiency; splenic rupture is a life-threatening emergency after abdominal trauma with massive splenomegaly, with no increased risk of spontaneous rupture in HS; and splenic sequestration, rare, presents as poorly tolerated acute anaemia, sometimes with shock, with sudden painful enlargement of the spleen, thrombocytopenia and neutropenia [8]. Iron overload without chronic transfusions or repeated parenteral iron is not usual in HS and should lead to reconsideration of a diagnosis of CDA and a search for cofactors, such as the HFE variant NM_000410.4:c.845G>A p.(Cys282Tyr) (C282Y) or variants in other haemochromatosis genes; in some patients no cofactor is found (section 7.5) [8, 68].

3.6. Pregnancy

In mild HS, anaemia worsens during pregnancy and returns to baseline after delivery; some women who have not undergone splenectomy develop anaemia severe enough to require transfusion [5, 6]. GeneReviews reports haemolytic crises and persistent anaemia during pregnancy in women with haemolytic anaemia, particularly those who have not undergone splenectomy, based on a series of 8 pregnant women [2]. In an Indian series of 10 pregnant women with HS followed between 2014 and 2020, anaemia, jaundice and weakness were frequent, most had a vaginal delivery at term, anaemia was more marked in those who had not undergone splenectomy, and neonatal HS was not identified; the series is small and lacks genetic confirmation [69]. The 2023–2024 parvovirus B19 epidemic was also associated with severe outcomes in pregnant women in Europe, according to the authors of the Czech study [66].

3.7. Gene-Specific Differences in Spherocytosis

Biallelic SPTA1 forms occupy the severe end of the spectrum, with transfusion dependence from fetal or neonatal life; the response to splenectomy depends on residual α-spectrin (section 2.3) [42]. EPB42-related HS is predominantly mild to moderate; children present with jaundice within the first 24 hours and, later, with haemolytic or aplastic crises associated with viral infections [2]. A patient homozygous for the null variant 4.2 Nancy, with no viable protein 4.2, developed a strong anti-protein 4.2 antibody response with alloimmune haemolytic anaemia after multiple transfusions for gastrointestinal bleeding; this phenomenon has not been described with other variants, although most patients with EPB42-related HS have no detectable membrane protein 4.2 [2]. SLC4A1-related HS tends to cause milder anaemia than the SPTA1 form [3]. Dominant ANK1- or SPTB-related HS is mild to moderate, with exceptions: some de novo variants, such as ankyrin Florianópolis, cause severe dominant HS [6].

3.8. Elliptocytosis, Pyropoikilocytosis and Ovalocytosis

HE has four clinical forms in the ICSH classification: asymptomatic carrier or non-haemolytic HE, with normal Hb and reticulocytes; HE with variable haemolysis; HE with transient infantile poikilocytosis; and HPP, with severe transfusion-dependent haemolytic anaemia from the neonatal period, Hb of 60–100 g/L and poikilocytosis. A single case of fatal hydrops fetalis due to homozygosity for a β-spectrin variant has been described [5]. Most heterozygotes have elliptocytes without fragmentation and do not require treatment; infections, such as CMV, may worsen haemolysis [3, 5]. In HPP, in vivo fragmentation is greater in the first year, and the percentage of microcytes is the best indicator of severity; erythrocytes show thermal instability [3, 5]. Severe HE and HPP share pigment gallstones, splenomegaly and aplastic crises with HS [3]. SAO is generally asymptomatic, with occasional mild haemolysis, jaundice or gallstones; it may be haemolytic in early childhood and haematologically normal in adulthood, and coinheritance with heterozygous α-thalassaemia does not worsen it [3, 5].

The French PNDS (National Diagnosis and Care Protocol) separates simple HE, heterozygous and almost always asymptomatic, with rare haemolytic episodes in situations of erythropoietic stress (bacterial, viral or parasitic infections, pregnancy, vitamin B12 deficiency), from persistent HPP, which is lifelong, with moderate (8–11 g/dL) or severe anaemia (Hb <8 and as low as <6 g/dL), and which usually results from the αLELY allele in trans with an SPTA1 variant or, more rarely, from homozygosity, compound heterozygosity or association with another red cell disorder; about 10% of patients with HE have a symptomatic form [8]. The “classic” West African form has more than 30% elliptocytes and sometimes low haptoglobin, without anaemia or haemolysis, but may become haemolytic in anaemia of another cause [8]. In SAO, the protocol reports a central retinal vein occlusion in a carrier with sickle cell trait and cases of iron overload with co-inherited β-thalassaemia trait [8].

3.9. PIEZO1-Related Xerocytosis and Gardos Channelopathy

The French retrospective series from Hôpital Bicêtre and the French hereditary stomatocytosis cohort included 126 patients from 64 families diagnosed between 1993 and 2017: 49 families with PIEZO1 and 6 families (12 patients) with KCNN4 [18]. The mean age at diagnosis of probands was 32±18 years (0–88), demonstrating the diagnostic delay. At diagnosis, 84% had chronic non-spherocytic haemolysis, 36% hyperferritinaemia, 11% thrombosis after splenectomy and 17% perinatal oedema; gallstones affected 52% (22% before age 20, 62% between 20 and 40 and 80% after 40) and splenomegaly 56% [18]. Mean Hb was 131±20 g/L, 68% were not anaemic, no patient depended on regular transfusions, and seven adults had Hb >160 g/L, two of whom were referred for suspected polycythaemia; mean MCV was 97±8 fL and MCHC 351±13 g/L, exceeding 360 g/L in 38% [18]. Plasma K+ was above the upper limit in 18 of 35 patients (51%), without specific testing for release at room temperature [18].

Iron overload is almost universal in xerocytosis, even without regular transfusions, and may cause organ dysfunction [70]. In the French series, mean ferritin at diagnosis was 764±480 ng/mL and increased with age (273±141 ng/mL before age 20, 717±441 between 20 and 40 and 1409±653 after 40), without a difference between carriers and non-carriers of HFE alleles; diabetes occurred in 3 patients, osteoporosis in 3, hypogonadism in 1, hypothyroidism in 1, and hepatocellular carcinoma led to liver transplantation in one patient [18]. The case published in 2025 of a man treated for years as having hereditary haemochromatosis illustrates the same situation (section 10) [71].

Perinatal oedema occurred in 22 patients from 13 families: hydrops (6), pleural effusion (5), ascites (11), hydramnios (2), pericardial effusion (2), subcutaneous oedema (3), hygroma (1) and jugular cyst (1); all those genotyped had PIEZO1, 13 had no known family history, there was 1 intrauterine death at 27 weeks, 1 medical termination at 28 weeks and 1 death at 15 days, and resolution was complete in 14 [18]. Perinatal oedema recurred in all informative families and is unrelated to fetal haemoglobin [3, 18]. Perinatal effusions usually regress but may progress to fatal hydrops [72].

Thrombosis is the severe complication of xerocytosis and is triggered predominantly by splenectomy: 12 of 16 splenectomised patients (75%) experienced 19 thrombotic events (5 portal vein thromboses, 5 strokes, 6 venous thromboembolic events, VTE, and 3 cases of chronic thromboembolic pulmonary hypertension), on average 13±8.6 years after surgery, and all 8 splenectomised patients with PIEZO1 developed thrombosis [18]. Thromboses were also described in four patients who had not undergone splenectomy [18].

Gardos channelopathy has a distinct phenotype. In the French series, patients with KCNN4 had lower Hb (102±13 versus 134±19 g/L; p<0.001), lower reticulocytes (178±63 versus 308±106 × 10⁹/L; p<0.001) and lower MCHC (332±13 versus 354±24 g/L; p<0.05) than patients with PIEZO1; 75% had Hb <120 g/L, compared with 27% of those with PIEZO1, stomatocytes were absent in 10 of 12, and ektacytometry was normal or atypical in the 6 families [18]. Adults with KCNN4 had higher ferritin (1702±1048 versus 656±428 ng/mL), and haemolysis persisted after splenectomy [18, 38]. The blood film shows non-specific abnormalities, with elliptocytes, schistocytes, hemighosts and rare stomatocytes [37]. Perinatal oedema had not been observed with KCNN4 until 2022, when a 28-bp deletion was associated with non-immune hydrops and fetal loss [73].

The French PNDS (National Diagnosis and Care Protocol), written with the participation of the same group, summarises the presenting forms of DHSt [8]. Haemolysis is almost constant but fluctuating, compensated in more than 70% of cases; outside the neonatal period, about 30% of patients have Hb below 12 g/dL and fewer than 10% below 10 g/dL, mainly with KCNN4, and anaemic forms are more frequent in the neonatal period, with transfusions sometimes needed in the first year. About 6% of adults have Hb at the upper limit of normal or above and are referred for polycythaemia, with stigmata of haemolysis and increased haemoglobin oxygen affinity (venous P50 at the lower limit or low); according to the protocol, DHSt would explain up to 10% of unexplained erythrocytoses and, in the few patients studied, red cell mass confirmed true polycythaemia [8]. In about 20% of cases there is a personal or family history of perinatal oedema, described mainly with PIEZO1 and attributed to the role of the protein in lymphatic valve development; it appears from the 2nd trimester, without concomitant fetal anaemia in most cases, may require drainage taps in utero or after birth, is associated with growth restriction and intrauterine fetal death, usually regresses in the first 6 months and does not recur in adulthood; in Gardos channelopathy, prenatal anaemia without oedema is described [8]. Hyperferritinaemia increases with age, independently of transfusions and genotype, and may be favoured by HFE variants; about two thirds of patients need chelation or iron depletion after the age of 40 years, and nearly 40% of asymptomatic relatives diagnosed through family screening already have hyperferritinaemia [8]. After splenectomy, thrombosis affects about 75% of patients, sometimes decades later, and haemolysis persists [8].

3.10. Overhydrated Stomatocytosis, Cryohydrocytosis, Pseudohyperkalaemia and Rh Deficiency

Source: ICSH 2015, table 3 [5]. DHSt prevalence differs between sources (section 9).

OHSt causes moderate to severe macrocytic haemolytic anaemia of fluctuating severity, with stomatocytes showing slit-like central pallor on the fixed blood film, splenomegaly or hepatosplenomegaly, cholelithiasis and a strong tendency towards iron overload independent of transfusions [3, 5]. Cryohydrocytosis has three forms: pseudohyperkalaemia alone; mild normocytic haemolysis with some stomatocytes; and the severe SLC2A1-related form, with marked haemolysis, pseudohyperkalaemia and paediatric neurological impairment [5]. Familial pseudohyperkalaemia is a trait with almost normal haematology, but falsely elevated K+ may lead to inappropriate treatments, such as fludrocortisone for presumed adrenal insufficiency, or to a mistaken diagnosis of Gordon syndrome in a French family with hypertension [5]. In regulator-type Rh deficiency syndrome, chronic haemolytic anaemia is usually compensated and mild to moderate, with stomatocytes and spherocytes, reticulocytosis and increased osmotic fragility; the predominant problem concerns transfusion, because anti-Rh29 alloimmunisation after transfusion or pregnancy requires the use of Rh-null units only and creates a risk of haemolytic disease of the fetus and newborn in subsequent pregnancies [3]. A healthy athlete with confirmed DHSt had haemolysis after every training session, an example of exercise-dependent expression [5].

3.11. Natural History and Treated Disease

The reference HS series combine patients with and without splenectomy, and interpretation of the figures requires the two situations to be distinguished. Splenectomy alters natural history: no aplastic crisis occurred after surgery in the Israeli cohort, splenectomy corrected anaemia in all protein subtypes in the Milan series and prevents the formation of new pigment stones, at the cost of a lifelong risk of severe infection and vascular events (sections 7.6 and 11) [6, 9, 61, 74]. In the Milan series, only 20% of patients underwent splenectomy between 1980 and 2005, and the rate decreased in the following decade [11]. In xerocytosis, the French series predominantly describes patients who had not undergone splenectomy, because splenectomy had been performed before the correct diagnosis, on average 15 years earlier, and did not abolish haemolysis [18].

4. Clinical Recognition and Warning Signs

4.1. When to Suspect the Diagnosis, by Age and Context

In the newborn, HS should be considered in the presence of early or marked jaundice with a negative DAT, particularly when mother and infant are ABO-incompatible but the DAT is negative, a situation in which another cause of haemolysis should be sought [65]. Jaundice in the first 24 hours with total serum bilirubin (TSB) above the phototherapy threshold probably indicates haemolytic disease, and identification of HS or another membrane defect helps predict recurrent haemolysis and risk in future pregnancies [75]. In prolonged neonatal jaundice (more than 2–4 weeks), haematological investigation includes a Coombs test, Rh and ABO compatibility and examination for abnormal erythrocytes such as spherocytes [43]. Indices suggesting HS in the newborn are MCHC >36.5–37 g/dL, MCHC/MCV ratio (HS index) >0.36 and increased RDW (red cell distribution width); the absence of spherocytes, family history, initial anaemia or reticulocytosis does not exclude the diagnosis [65]. An MCHC >360 g/L in the newborn had 82% sensitivity and 98% specificity for HS in the study cited by the British guideline [6].

In infants and children, suspicion arises in the presence of anaemia with reticulocytosis that does not respond to iron, intermittent jaundice, splenomegaly, gallstones before adolescence or an aplastic crisis as the first manifestation [2, 9, 66]. In adults, mild HS is recognised through fluctuating unconjugated jaundice, gallstones at a young age, splenomegaly discovered on imaging, worsening anaemia during pregnancy or infection, or investigation of a relative [2, 5]. Family history is central: approximately 75% of patients have an affected parent, but its absence does not exclude the disease, owing to recessive inheritance, a de novo variant or undiagnosed mild disease in the parent [2, 6].

4.2. Clues to a Membrane Disorder Other Than Spherocytosis

Certain features point to another entity and should be sought before any decision to perform splenectomy. An MCV >100 fL in a patient with “HS” should raise suspicion of stomatocytosis [6]. In xerocytosis, MCHC is high and osmotic fragility reduced, the opposite of what is expected in HS; haemolysis is often compensated, with normal or even high Hb, and iron overload, pseudohyperkalaemia and a history of perinatal oedema or effusions are strong clues [3, 18]. A child with xerocytosis was treated for iron deficiency for three years before diagnosis by exome sequencing [3]. Pseudohyperkalaemia is recognised when K+ is high in a sample left at room temperature but normal in a sample separated immediately [3]. Failure to respond to splenectomy performed for “HS” should prompt diagnostic reassessment for stomatocytosis or type II CDA [5, 6]. Bizarre morphology with MCV <70 fL suggests HPP or the coexistence of two diseases and warrants family investigation [5]. Southeast Asian or Melanesian origin with rigid ovalocytes suggests SAO; the combination of haemolytic anaemia and hyperchloraemic metabolic acidosis with an inappropriately high urinary pH suggests SLC4A1-related dRTA [5, 23]. Spherocytosis with dysmorphism or intellectual disability suggests an 8p11.2 deletion [5].

4.3. Warning Signs and Emergencies

4.4. Newborn Screening

The sources consulted do not describe population newborn screening programmes for HS or other membrane disorders. Neonatal surveillance is targeted: the newborn of a parent with HS should have bilirubin monitored for several days during the first week and Hb during the first 2–4 months [2, 6]. The Thai study of 1505 newborns screened using cord blood for three founder SPTB variants is a research study; it showed a prevalence of 0.86% and demonstrated that most carriers had neither anaemia nor a diagnosis at birth, supporting bilirubin surveillance in a newborn with a known SPTB variant even with a normal blood count [3, 44].

5. Diagnosis: Criteria, Tests and Algorithms

5.1. British Guideline Criteria

The British Society for Haematology guideline (BSH, then the British Committee for Standards in Haematology), updated in 2011 and published in 2012 with GRADE grading (Grading of Recommendations Assessment, Development and Evaluation; grade 1 strong, grade 2 weak; evidence A high, B moderate, C low), remains the guideline for HS diagnosis and treatment; the sources consulted did not identify a subsequent replacement [6]. Patients with a family history of HS, typical clinical findings (splenomegaly) and typical laboratory findings (spherocytes, increased MCHC, reticulocytosis) do not require additional tests (grade 1, evidence A). If the diagnosis is equivocal, for example with few spherocytes and no other findings, a screening test with high predictive value, cryohaemolysis or EMA binding, is recommended (grade 1, evidence A), followed by family investigation if the result remains equivocal. Membrane protein electrophoresis by SDS-PAGE is the method of choice to confirm selected cases with equivocal or borderline screening results: when the clinical phenotype is more severe than morphology predicts, when the patient's morphology is more severe than that of affected parents, and when the diagnosis is unclear before splenectomy and stomatocytosis is possible (grade 1, evidence B) [6]. In 2011, the guideline stated that the diagnosis of HS does not require molecular analysis [6]; indications for genetic testing have since expanded (section 5.7) [5, 10].

The 2021 French PNDS (National Diagnosis and Care Protocol), with the HAS grades (A, established scientific evidence; B, scientific presumption; C, low level of evidence) and consensus categories 1 to 3, recommends the EMA test as the first-line screening or confirmatory test, with concordant morphology on the blood film (grade A, category 1), and accepts it in the neonatal period when a parent has HS [8]. Some French teams accept the diagnosis without further tests in the presence of a family history and a typical picture, with HS morphology, normal MCV and MCHC >36%; without a family history, the aetiological work-up includes the DAT, haemoglobin fraction analysis, red cell G6PD, the blood film and EMA [8]. These tests are interpretable only more than 2 months after a transfusion, an interval close to the 8 weeks of the ICSH [5, 8]. In first-degree relatives of a patient with documented HS, the protocol calls for a full blood count with reticulocytes, haptoglobin, LDH and bilirubin, completed with EMA if there is haemolysis and, in adults, with full iron studies [8].

5.2. Full Blood Count, Red Cell Indices and Blood Film

The laboratory pattern is that of non-immune haemolytic anaemia: reticulocytosis (2.5% to over 10%, normal or low during an aplastic crisis), low or undetectable haptoglobin, increased lactate dehydrogenase (LDH) and unconjugated bilirubin, and a negative DAT [2, 5]. Normal haptoglobin after 6 months of age ranges from 16 to 200 mg/dL; in HS it is typically undetectable but may normalise during concomitant inflammation because it is an acute-phase reactant [2]. Normal MCHC ranges from 31 to 37 g/dL; in HS it is generally between 35.5 and 37.5 g/dL [2]. A DAT should always be performed in newly diagnosed haemolytic anaemia because autoimmune haemolytic anaemia (AIHA) has the same morphology [2, 3].

Modern analysers provide indices with diagnostic value. The percentage of hyperdense cells (cellular Hb >410 g/L, Advia analyser) is predictive of HS when the family history and indices are compatible, particularly if ≥4%, but is also increased in infantile pyknocytosis, immune spherocytosis, DHSt and CDA II [5]. A difference between MCV and mean spherical cell volume (MSCV, Beckman Coulter analysers) >9.6 fL had 90.6% specificity and 100% sensitivity for DAT-negative HS [5]. In the Sysmex algorithm, a high ratio of reticulocytes to immature reticulocyte fraction (>19) detects mild or subclinical HS with 100% sensitivity, and the combination of increased microcytes and low hypochromic reticulocyte haemoglobin detects moderate and severe forms [5]. MCV does not discriminate HS [5]. Reticulocyte volume is <100 fL in HS, except in newborns [5].

Spherocytes predominate on the blood film; in mild HS they may be occasional or appear only during a crisis, and “mushroom” cells are associated with band 3 defects [5]. Some ovalocytes and elliptocytes may be present [2]. Spherocytes on the blood film have good positive predictive value when the family history and indices are compatible; without a family history, a DAT and a screening test should be performed [5]. Mild HS, with occasional spherocytes, borderline reticulocytosis and slightly abnormal haemolysis markers, is diagnosed only after excluding pregnancy, AIHA, CDA II, systemic haematological diseases and certain enzyme deficiencies [5]. The ICSH recommends that the request include the provisional diagnosis, clinical findings, DAT result, reticulocytes, haemolysis markers and family history, and that the sample be obtained before transfusion or after clearance of transfused erythrocytes, namely 8 weeks after transfusion [5].

5.3. Membrane Screening Tests

The EMA test uses flow cytometry to measure the fluorescence of erythrocytes labelled with eosin-5′-maleimide, which binds to band 3, Rh, RhAG and CD47; in HS, membrane loss reduces fluorescence [78]. The result is obtained in 2–3 hours and is positive regardless of morphology and age, making it useful in newborns [6]. The acidified glycerol lysis test (AGLT) measures the time to 50% haemolysis in a hypotonic glycerol solution acidified to pH 6.85; normal is >900 s [5]. Classical osmotic fragility (OF) testing uses 14 NaCl concentrations from 0.1 to 0.8 g/dL, with or without preincubation for 24 hours at 37 °C, which increases lysis [5]. Cryohaemolysis measures lysis after cooling [6]. Osmotic gradient ektacytometry measures the deformability index under shear stress across an osmolality gradient and defines three parameters: Omin, the osmolality at which the index is minimal, corresponding to the 50% haemolysis point of OF and dependent on the surface-to-volume ratio; EImax or DImax, maximal elongation, related to the mechanical properties of the skeleton; and Ohyper, the osmolality at which the index falls to half on the hypertonic limb, which decreases when intracellular viscosity increases [2, 5].

Sources: [5, 61, 78–80].

Sensitivities differ between studies because the populations differ: series of consecutive patients from reference centres with an established diagnosis, such as the Milan series, give higher values than a small series compared with healthy controls, such as the Barcelona series [78, 80]. Normal OF occurs in 10–20% of patients with HS, and the British guideline has not recommended it for routine use since 2004 [6]. All screening tests detect typical HS; when in doubt, two should be used [5]. No single test detects all cases [78]. The French PNDS (National Diagnosis and Care Protocol) places the threshold for reduced EMA binding between 11% and 21%, depending on the laboratory, for lack of consensus, and sensitivity and specificity in the range of 90–99%; the pre-analytical conditions (transport at room temperature, stability of at least 72 hours, small sample volume) make it suitable as a first-line test, available in outpatient laboratories in France [8]. Osmotic resistance tests (manual, Pink test, flow cytometry) have moderate sensitivity and should not be used alone for initial screening; haptoglobin is the most sensitive haemolysis marker and is consistently low in HS [8].

False-positive and false-negative results have known causes. OF is positive in immune anaemia, after recent transfusion, in enzyme deficiencies (G6PD, pyruvate kinase, PK) and in unstable haemoglobins [5]. AGLT is falsely positive in AIHA, in approximately one-third of pregnant women, in chronic renal failure and in myelodysplastic syndromes [5]. EMA is negative in all AIHA, even with marked spherocytosis, but also detects SAO, cryohydrocytosis and some cases of CDA II; in HPP, fluorescence is ~25% lower than in HS, and macrocytic erythrocytes (MCV ≥100 fL) yield fluorescence above the reference range, as in megaloblastic anaemia, OHSt, DHSt, erythrocytes coated with cold agglutinins, CDA I and PK deficiency [5, 6, 78]. EMA reference ranges are specific to each laboratory and cytometer, and the lack of harmonisation necessitates family investigation when a result falls within a grey zone [5]. Ektacytometry should be performed on the day of collection (up to overnight storage at 4 °C; more than 24 hours simulates overhydration), requires a negative DAT, is unreliable after recent transfusion and is available in few centres [3, 5].

Sources: BSH 2011, table III, and ICSH 2015, section 6.2.3 [5, 6]. Ektacytometry is the only simple, reliable screening test for DHSt [5].

Genotype-based analyses show group differences. In the Utrecht series, hydration was more affected in patients with SLC4A1 (median Ohyper 416 mOsm/kg), Omin width was greater with SPTB and ANK1, and the parameters distinguished mild from moderate HS [7]. In the Milan series, the HS2 profile was associated with positive OF in fresh blood in 82.3%, compared with 60.1% for the HS1 profile (p=0.007), without differences in EMA [40].

5.4. Membrane Protein Electrophoresis

SDS-PAGE quantifies membrane proteins and shows the deficiencies of HS and HE, increased spectrin dimers in HE and HPP, hypoglycosylated compact band 3 in CDA II and spectrin variants; it does not detect GLUT1, RhAG or PIEZO1 [5, 6]. In HS, 8–11% of patients have no detectable protein reduction, according to data from Bristol, Milan and Naples [5]. Reticulocytosis masks ankyrin deficiency, which SDS-PAGE does not detect in membranes from patients who have not undergone splenectomy; after splenectomy, the defect was identified in all previously unclassified patients in the Milan series [6, 61]. Isolated protein 4.2 deficiency may result from EPB42 or SLC4A1 variants, and SDS-PAGE does not establish the gene involved [3, 5]. Its main limitation is lack of sensitivity in very mild HS [6].

5.5. Tests for Stomatocytoses

A firm diagnosis of stomatocytosis is important because splenectomy benefits neither DHSt nor OHSt [5]. Confirmation uses ion flux measurements, ektacytometry and sequencing [5]. Where these tests are unavailable, flame photometry measures plasma K+ and intracellular K+ and Na+ concentrations in fresh samples and samples stored at room temperature and refrigerated, with a matched control [5]. Cryohydrocytosis can be confirmed by sequencing the C-terminal domain of SLC4A1 [5]. To distinguish PIEZO1 from KCNN4, reticulocyte indices from the ADVIA 2120 analyser are useful: in Gardos channelopathy, reticulocyte MCV is lower than overall MCV and reticulocyte MCHC higher than MCHC, the reverse of normal and of PIEZO1 xerocytosis; the difference (reticulocyte MCV − MCV) was −0.9±5 fL in 15 KCNN4 patients versus 19.8±3 fL in 79 PIEZO1 patients [81]. GeneGuidance reports ΔMCV thresholds of 8.6 fL and ΔMCHC thresholds of −5.5 g/L with 100% sensitivity and specificity in that series; the index provides guidance, but confirmation requires identification of the variant [3, 81]. Because ektacytometry is normal or atypical in Gardos channelopathy, genetic testing is necessary even with normal ektacytometry [18]. Sitosterolaemia causes mild haemolysis with very marked stomatocytosis without cation leakage and is distinguished by macrothrombocytopenia (mean platelet volume ~10–15 fL) and measurement of plasma sterols by mass spectrometry [5, 67].

In the French PNDS (National Diagnosis and Care Protocol), normal haemolysis markers, including haptoglobin, do not exclude DHSt; anaemia is normocytic or macrocytic, the DAT, haemoglobin fractions, G6PD, PK and EMA are classically normal, SDS-PAGE is normal and of no value, and genetic testing is recommended in all cases (PIEZO1, KCNN4, SLC4A1), all the more so with normal or inconclusive ektacytometry, because it defines the type, the prognosis and the contraindication to splenectomy; in adults, the work-up includes iron, transferrin and ferritin [8]. Family screening includes full blood count, reticulocytes, blood film, haemolysis markers, iron studies and genetics or ektacytometry, and a search for genetic factors of iron overload when overload is present [8].

5.6. Elliptocytosis and Pyropoikilocytosis

Ektacytometry and morphology provide the phenotypic basis for diagnosing HE; ektacytometry does not predict severity, and morphology does not distinguish the genes [3]. Without a family history and with few elliptocytes, diagnosis should be confirmed by SDS-PAGE (protein 4.1, spectrin dimers, variants) or ektacytometry [5]. In HPP, testing for the αLELY allele by PCR explains the severe phenotype in most patients; the thermal stability test is characteristic [3, 5]. EMA has been validated predominantly for HS [3]. Exclusions include iron deficiency, thalassaemia, acquired elliptocytosis (burn patients, myelodysplasia, heated samples) and the Leach phenotype (Gerbich-null), in healthy individuals with elliptocytes and a 20% reduction in protein 4.1 [5].

In symptomatic HE, the French PNDS (National Diagnosis and Care Protocol) notes a spurious microcytosis due to fragmentation, which alters the analyser indices, normal MCHC and an EMA result that is normal or shows fluorescence reduced in proportion to fragmentation, in a dual population; ektacytometry is the test of choice in symptomatic forms, especially if splenectomy is being discussed or in a family study, and SDS-PAGE identifies the affected protein in severe forms [8]. The protocol reserves genetics for severe forms (HPP), regularly transfused patients in whom phenotyping is not possible, associations with other red cell disorders and the rare severe or in utero lethal forms that may justify prenatal diagnosis, and adds testing for Gilbert syndrome by analysis of the UGT1A1 promoter [8]. Family screening is recommended in symptomatic HE and when one member of a couple has simple HE or SAO, with full blood count, reticulocytes, haptoglobin, LDH, bilirubin, blood film and ferritin [8].

5.7. Molecular Tests

The ICSH reserves molecular analysis for selected cases: a proband with features of a membrane disorder and negative or equivocal screening tests in a haematologically normal family, suspected recessive inheritance, a de novo variant or compound heterozygosity, and decision-making regarding splenectomy; it adds no information in a family with an established diagnosis [5]. Molecular testing of all relatives is useful when the proband's phenotype is more severe than that of affected relatives, because it reveals recessive inheritance or a de novo variant [3]. Next-generation sequencing (NGS) with panels including coding and non-coding variants, or exome and genome sequencing, improves diagnostic accuracy and speed, particularly in severe transfusion-dependent cases in which phenotyping is difficult; after recent transfusions and when functional tests are unavailable, it is often the only route [10, 40]. Neither EMA nor electrophoresis identifies the gene, and differences in inheritance and recurrence risk between genes justify sequencing when counselling depends on it [3].

Sources: [5, 59, 60, 82].

These figures are yields in selected series and do not equate to the clinical sensitivity of testing in the patient population. For EPB42, sequencing detects all known pathogenic variants, and the only reported deletion (32 bp) is detectable by sequencing; this proportion of detectable variants is not clinical sensitivity [2]. Technical limitations are known: NGS has difficulty with deep intronic and regulatory variants and deletions, which matters particularly in recessive inheritance [5]; deep intronic ANK1 variants were found only through genome sequencing and functional investigation [41]; the αLEPRA allele is intronic and should be specifically sought when the proband's phenotype is more severe than expected [5]; and variants in more than one gene may coexist in the same patient [5]. A positive result does not establish causality, and genotype–phenotype correlation is essential [10]. A negative result does not exclude the disease.

The report should state the gene, reference transcript with version (for example NM_000342.4 for SLC4A1 or NM_001114134.2 for EPB42), HGVS variant at DNA and protein levels, zygosity and classification according to American College of Medical Genetics and Genomics and Association for Molecular Pathology (ACMG/AMP) criteria [12, 45]. Variants of uncertain significance (VUS) are frequent in membrane genes: ClinVar recorded 292 pathogenic variants, 313 likely pathogenic variants and 709 VUS in ANK1, figures reflecting the breadth of panels rather than disease frequency [3]. Interpretation of a VUS requires phenotyping (EMA, ektacytometry, SDS-PAGE), family segregation and, when available, functional investigation, such as expression in Xenopus oocytes [5, 24]. In the case in section 10, the patient's PIEZO1 variant is classified as a VUS in ClinVar, and the authors argue for its pathogenicity on the basis of the phenotype [53, 71].

In France, NGS panels are declared to the Agence de la biomédecine (ANPGM_137 reference framework for the molecular diagnosis of red cell disorders) and performed in accredited laboratories [8]. The PNDS restricts the indications in HS to recessive or sporadic forms, transfusion-dependent patients with falsified phenotypic tests, complex or mixed forms (for example, thalassaemia minor masking spherocytosis), exceptional cases of prenatal diagnosis in very severe forms and unexplained fetal anaemia or hydrops; it also recommends testing for Gilbert syndrome, which calls for closer surveillance for gallstones, and explanation of the results in a genetics clinic, with family screening [8]. The panel in the French algorithm is a “constitutional haemolysis” panel that includes at least the genes for membrane proteins and red cell enzymes [8].

5.8. Diagnostic Algorithm

In chronic haemolysis with a negative DAT and spherocytes, the algorithm proposed by Bianchi and colleagues combines EMA and AGLT: if both are negative, HS is excluded; a positive EMA confirms HS, except in non-dominant cases with inadequate reticulocytosis, in which SDS-PAGE should exclude CDA II; and a negative EMA with a positive AGLT without a family history leads to SDS-PAGE [78]. In the same series, 13% of cases referred as HS were CDA II [78]. The practical pathway is as follows: full blood count, reticulocytes, blood film, haemolysis markers and DAT; with a family history and a typical presentation, the diagnosis is established; without a family history or with an atypical presentation, one or two screening tests; if discordant or if features of stomatocytosis are present (high MCV, high MCHC with reduced OF, pseudohyperkalaemia, high ferritin), ektacytometry and cation flux measurements; and, in severe, recessive or transfused cases, or before splenectomy if uncertainty remains, a molecular panel [5, 6, 10]. In a clinically stable newborn, tests may be deferred until 6 months, although EMA is interpretable at any age [6]. Relatives (parents and siblings) should be investigated with a full blood count, reticulocytes, blood film and EMA or ektacytometry, and testing for familial variants when known [2, 5].

The French PNDS (National Diagnosis and Care Protocol) algorithm for suspected HS starts from the family history [8]. With a family history and a typical profile (HS morphology, normal MCV, reticulocytes >150 × 10⁹/L, MCHC >36%), the diagnosis can be accepted or confirmed by a reduced EMA; if EMA is not reduced, ektacytometry decides. Without a family history, a negative DAT, normal haemoglobin electrophoresis and normal G6PD and PK are required first; with a typical profile, a reduced EMA confirms HS and, if it is not reduced, a positive ektacytometry confirms it and a negative one leads to molecular testing. In an atypical picture (non-typical morphology, abnormal MCV, reticulocytes <150 × 10⁹/L or iron overload), EMA and ektacytometry are performed: both positive confirm HS, both negative point to another diagnosis, and discordant results lead to molecular testing, with or without SDS-PAGE [8]. The general approach to haemolysis in the same protocol includes, whenever possible, the full blood count, reticulocytes, LDH and haptoglobin of both parents, and recalls the acquired membrane disorders (paroxysmal nocturnal haemoglobinuria, autoimmune lymphoproliferative syndrome, elliptocytosis of myelodysplasia) and neonatal pyknocytosis [8].

5.9. Prenatal and Preimplantation Diagnosis

Prenatal diagnosis and preimplantation genetic testing are possible when the familial variants have been identified [2]. They are mainly of interest in severe recessive forms, such as HS due to biallelic SPTA1 with a risk of hydrops [42, 64]. In fetal surveillance of at-risk pregnancies, middle cerebral artery (MCA) Doppler detects fetal anaemia but does not by itself predict postnatal severity; the decision integrates the maternal and family history, genetics and serial surveillance [64]. When one of the parents has xerocytosis, ultrasound surveillance at least monthly aims to detect perinatal oedema and effusions [18]. The French PNDS (National Diagnosis and Care Protocol) considers prenatal diagnosis exceptional, reserved for very severe transfusion-dependent forms or those with a risk of death in utero, unexplained fetal anaemia and hydrops [8].

6. Differential Diagnosis

6.1. Comparative Table

Sources: [2, 3, 5, 6, 23, 63, 81, 83].

6.2. Main Differential Diagnoses

AIHA is the main differential diagnosis of HS without a family history. It is rare in children, generally post-viral and transient, and is usually, but not always, excluded by a negative DAT [6]. Ektacytometry does not distinguish it from HS because antibodies alter the parameters, making a negative DAT a prerequisite for the test; EMA, conversely, is negative in all AIHA [5, 78]. In a jaundiced newborn, ABO incompatibility with a positive DAT is the main differential diagnosis; with a negative DAT, another cause should be sought [65]. Coombs testing, Rh and ABO are part of the initial investigation [43].

CDA type II is the most treacherous differential: mild haemolytic anaemia, jaundice, splenomegaly, microspherocytes and increased OF mimic HS, and several patients have been splenectomised without remission [5, 6]. Recessive inheritance, disproportionately low reticulocytosis (below 150 × 10⁹/L at the 90th percentile of most CDA II; a suspected HS with reticulocytes of 120–180 × 10⁹/L requires differentiation), poikilocytosis, compact band 3 on SDS-PAGE, binucleated erythroblasts in the marrow and iron overload disproportionate to transfusions separate the two disorders; confirmation is by SEC23B sequencing [2, 5, 6].

Enzymopathies (PK, G6PD) are distinguished by the absence of spherocytes and normal OF and ektacytometry; some, such as triosephosphate isomerase (TPI) and phosphoglycerate kinase (PGK1) deficiencies, include neurological and muscular manifestations [2, 83]. Initial assessment of any haemolytic anaemia includes a full blood count with reticulocytes, blood film review, DAT and indirect antiglobulin testing, haemoglobin electrophoresis and G6PD activity, particularly in males [2]. HS is readily distinguished from HbH disease and β-thalassaemia, which feature microcytosis and hypochromia, but coinheritance of β-thalassaemia trait or sickle cell disease confounds the presentation, and iron, folate or vitamin B12 deficiencies mask laboratory findings; obstructive jaundice alters membrane lipids, masks morphology and reduces haemolysis [2, 6]. Infantile pyknocytosis predominantly affects preterm infants, causes transient haemolytic anaemia with reticulocytosis and hyperbilirubinaemia, may require transfusion at 6–9 months and resolves [5, 6].

The distinction between HS and stomatocytosis has the greatest therapeutic consequences, because splenectomy is effective in the former and dangerous in the latter [2, 11]. Homozygotes for the EPB42 variants 4.2 Nippon and 4.2 Komatsu have ovalocytes and stomatocytes on the blood film, and GeneReviews emphasises the need to exclude stomatocytosis before splenectomy [2]. Xerocytosis is distinguished by high MCHC with reduced OF, normal or high Hb, pseudohyperkalaemia, elevated ferritin and a left-shifted ektacytometry curve [5, 18]. In an Argentinian series of 20 children with DHSt, elevated MCHC with lysis-resistant erythrocytes pointed to PIEZO1, and the absence of clear dehydration to KCNN4; macrocytosis, haemolytic anaemia and iron overload were common to both forms [84]. Normal ektacytometry does not exclude Gardos channelopathy, and reticulocyte indices guide gene selection [18, 81]. Sitosterolaemia mimics hereditary stomatocytosis and is distinguished by macrothrombocytopenia [5, 67].

Acquired membrane disorders complete the differential: AIHA produces many spherocytes and can falsify some functional tests; the protease inhibitors used in hepatitis C can give a picture of elliptocytosis or spherocytosis; the oxidative stress of the enzymopathies damages the membrane–skeleton complex; the acquired protein 4.1R deficiency of myelodysplastic syndromes, with or without a tumour karyotype abnormality, gives more than 10% elliptocytes; and an acquired stomatocytosis occurs in alcoholism and with vincristine or vinblastine [8].

In syndromic forms, spherocytosis associated with dysmorphism, intellectual disability or neurological abnormalities prompts microarray testing for an 8p11.2 deletion including ANK1 [3, 5]. Elliptocytosis associated with Alport nephropathy, deafness, intellectual disability and midfacial hypoplasia in a boy suggests a contiguous Xq22.3 deletion involving COL4A5 and ACSL4 [3, 63]. Haemolytic anaemia with distal renal tubular acidosis, particularly in Southeast Asia, the Middle East or India, suggests biallelic SLC4A1 variants; in a series of 78 patients with SLC4A1-related dRTA and anaemia, Hb ranged from 4.4 to 15.7 g/dL, and alkali therapy was associated with correction of anaemia and reticulocytosis [23].

7. Treatments and Risk Management Strategies

7.1. Principles and Initial Assessment

There is no approved targeted pharmacological therapy for HS or other membrane disorders; treatment is supportive and surgical, and its intensity follows clinical severity rather than the gene [3, 6]. After diagnosis, GeneReviews recommends measuring bilirubin, assessing severity through Hb, reticulocytes and transfusion history, measuring ferritin, performing abdominal ultrasonography for gallstones when symptoms are present or from age 10–12 years in patients with significant haemolysis, assessing spleen size by ultrasonography when physical examination is inconclusive or contact sports are planned, and offering genetic counselling [2]. In children, an annual appointment is sufficient once baseline status has been established; without symptoms, a full blood count at every appointment is unnecessary, but growth should be monitored and parents informed of the risk of sudden anaemia due to parvovirus [6].

The French PNDS (National Diagnosis and Care Protocol) defines the initial work-up for HS: full blood count with reticulocytes, blood film, total and indirect bilirubin, LDH, haptoglobin, blood group with Rh-Kell phenotype, irregular antibody screen and EMA; serology for human immunodeficiency virus (HIV) and hepatitis C and B, which is no longer systematic in transfusion follow-up but forms part of the initial work-up; parvovirus B19 serology from 1 year; baseline abdominal ultrasound (gallbladder, spleen size and volume) from 3 years, or earlier if there is splenomegaly; and, without a family history, study of both parents with haemolysis markers, blood film and EMA [8]. The aims of follow-up are to assess the tolerance of chronic anaemia, screen for gallstones, splenomegaly, folate deficiency and iron overload, discuss splenectomy and cholecystectomy and update genetic counselling across generations; a consultation in a specialised centre is offered in symptomatic forms [8].

7.2. Neonatal Hyperbilirubinaemia

The 2022 American Academy of Pediatrics (AAP) guideline for newborns at 35 or more weeks of gestation replaces the 2004 guideline still cited by GeneReviews [2, 75]. It classifies gestational age <38 weeks, albumin <3.0 g/dL, isoimmune haemolytic disease, G6PD deficiency or “other haemolytic conditions”, sepsis and significant clinical instability in the preceding 24 hours as risk factors for bilirubin neurotoxicity; their presence lowers thresholds for phototherapy and escalation of care [75]. Non-immune haemolysis has not been demonstrated to increase neurotoxicity risk, but the guideline considers it prudent to assume that it does [75]. Intensive phototherapy starts at thresholds defined by gestational age, risk factors and age in hours; TSB should be repeated within 12 hours of initiation; phototherapy may be discontinued when TSB falls at least 2 mg/dL below the initiation threshold, and a longer duration is an option when rebound risk is present, for example in haemolytic disease [75]. Genomic sequencing may help when the cause of haemolysis remains unidentified in a newborn requiring escalation of care [75]. Conjugated hyperbilirubinaemia requires assessment for cholestasis; in obstructive jaundice due to excessive haemolysis, GeneReviews suggests considering hypertransfusion to suppress endogenous erythropoiesis [2].

The French PNDS (National Diagnosis and Care Protocol) organises the management of neonatal jaundice in three stages [8]. The first is systematic screening, with the risk factors for severe hyperbilirubinaemia (jaundice before 24 hours, gestational age <38 weeks, African, Caribbean or Asian family origin, ineffective breastfeeding) and blood or transcutaneous measurement compared with hour-specific reference curves. The second is prompt treatment by phototherapy, exchange transfusion or pharmacological agents, haemolytic hyperbilirubinaemia being an emergency because it progresses quickly. The third is individualised follow-up in the first 15 days, with transmission of information from the maternity unit, information for parents, recognition of the signs of severe hyperbilirubinaemia (inconsolable or drowsy newborn, high-pitched cry, neck hyperextension, head retroflexion), coordination with the care network after discharge and delayed discharge if surveillance cannot be ensured [8].

7.3. Folic Acid

Chronic haemolysis increases folate requirements. The British guideline recommends supplementation in moderate and severe HS and considers it unnecessary in mild HS, without specifying a dose [6]. GeneReviews proposes 400 µg once daily during the first year and 1 mg once daily thereafter, and 800–1000 µg daily during pregnancy; the sources consulted did not identify a primary source for this regimen [2]. The US label for folic acid tablets specifies a usual therapeutic dose of up to 1 mg daily in adults and children regardless of age, allows higher maintenance doses in haemolytic anaemia, and cautions that doses above 1 mg daily do not increase the haematological effect and that doses above 0.1 mg should be given only after vitamin B12 deficiency has been excluded; the labelled indication is megaloblastic anaemia due to folate deficiency, not haemolytic anaemia [85].

Folate doses differ about fivefold between sources: the French PNDS proposes 2.5–5 mg daily (or 3 times a week), GeneReviews 0.4–1 mg daily, and the US label considers that doses above 1 mg daily do not increase the haematological effect; in the absence of comparative trials, the national guideline dose is the reference and the label serves for comparison [2, 8, 85]. The PNDS considers the supplement probably unnecessary in minor HS and, in DHSt, advises against any iron intake outside true deficiency, including vitamin supplements [8].

7.4. Transfusion and Erythropoietin

Red cell concentrate transfusions treat aplastic and haemolytic crises and the severe anaemia of the first year [2]. In the Czech study, transfusion was given when Hb was below 7.5 g/dL, with a median of 2 units per child, and discharge when Hb was above 7.5 g/dL with stability [66]. In the Israeli cohort, patients with higher baseline reticulocytes needed more transfusions during the aplastic crisis, which suggests closer surveillance of severe phenotypes [9]. In infants, the British guideline recommends avoiding repeated transfusions when possible; erythropoietin may reduce or avoid transfusions in the first year and is usually stopped at 9 months, but the guideline gives no dose; the French PNDS (National Diagnosis and Care Protocol) regimen is in the following table [6]. Regularly transfused patients should receive genotyped red cells [6]. In Rh deficiency syndrome, only Rh-null units can be used, obtained from compatible relatives, frozen inventories or rare donor registries; autologous donation and cryopreservation should be considered [3]. In EPB42-related HS with a null allele, anti-protein 4.2 alloimmunisation after multiple transfusions has been described in one patient [2].

The French PNDS sets the neonatal transfusion threshold, for newborns of more than 32 weeks and 1500 g, at Hb <7 g/dL with reticulocytes <100 × 10⁹/L without clinical signs, but accepts transfusing above 7 g/dL if tolerance is poor, because the fall is rapid in the first month; the volume is 15–20 mL/kg of phenotyped red cell concentrate, at a maximum rate of 5 mL/kg/h, and crossmatching is useful only with maternal–fetal alloimmunisation (positive irregular antibody screen) [8]. Outside the neonatal period, the transfusion target in children is at least the Hb corresponding to −2 standard deviations for age; in adults, transfusion is indicated for poorly tolerated anaemia below 6 g/dL, with a threshold of 8 or even 10 g/dL in heart disease; in the protocol's summary sheet, the hyperhaemolysis of an infection prompts discussion of transfusion below 7 g/dL [8]. Transfusions, phenotyped for the Rh and Kell systems, are given on demand during exacerbations or on a regular schedule when chronic anaemia is very severe or poorly tolerated, with iron monitoring and discussion of splenectomy [8].

Erythropoietin divides the sources: the PNDS acknowledges the lack of high-level evidence, limits treatment to the first 3 months, because endogenous erythropoietin rises quickly after the neonatal period, and combines it with iron; the British guideline usually stops it at 9 months, without giving a dose; and the PNDS weekly dose of epoetin beta is about 1.3 times that of the SmPC for prematurity, the only approved neonatal indication [6, 8, 86].

7.5. Iron and Chelation

Supplemental iron should be given only for documented iron deficiency, with monitoring of ferritin and transferrin saturation and discontinuation after repletion [2]. The PNDS (National Diagnosis and Care Protocol) adds that iron deficiency is rare in HS and more likely in infants, pregnant women and adolescent girls with menorrhagia [8]. In a child who remains transfusion-dependent after the first year, GeneReviews recommends strongly considering chelation, and liver T2* magnetic resonance imaging (MRI) when ferritin remains above 300–500 ng/mL [2]. The French PNDS recommends systematic screening for iron overload in adults with HS, with ferritin, transferrin saturation and serum iron (grade B, category 2A), because ferritin alone is altered by haemolysis; with ferritin >500 µg/L and/or transferrin saturation >50% in men or >45% in women without explanation, liver MRI quantifies iron (grade B, category 2A), and an LIC >100 µmol/g dry liver leads to discussion of treatment [8]. The protocol gives the equivalence of 7 mg/g for this threshold in HS and 5–7 mg/g in DHSt; the stoichiometric conversion of 100 µmol/g gives about 5.6 mg/g [8]. When genetic factors are found, family screening is necessary [8]. In the stomatocytoses, iron overload should be monitored regardless of transfusions [3, 70]. In the French xerocytosis series, patients were treated by phlebotomy (15), deferasirox (9), desferrioxamine (3) or deferiprone (1), with a fall in liver iron concentration (LIC) from 200±103 µmol/g at diagnosis to 88±42 µmol/g at last follow-up (p<0.001); the source does not give doses [18]. Ferritin correlates poorly with LIC below 1000 ng/mL: eight patients with ferritin below 1000 ng/mL already had LIC above 150 µmol/g [18]. Phlebotomy is possible in patients with preserved Hb, as in the case in section 10 [71].

Deferasirox is the oral chelator for which the most labelling data were extracted. In the USA, it is indicated for chronic transfusional iron overload in patients aged 2 years or older and for iron overload in non-transfusion-dependent thalassaemia (NTDT) syndromes from age 10 years, with LIC ≥5 mg Fe/g dry weight and ferritin >300 µg/L [88]. It should be initiated only with evidence of transfusional overload, namely at least 100 mL/kg of red cell concentrate (approximately 20 units in a 40-kg patient) and ferritin consistently above 1000 µg/L [88].

In the European Union, Exjade® (authorised on 28 August 2006) is indicated first line in transfused β-thalassaemia major from age 6 years and, when desferrioxamine is contraindicated or inadequate, in patients with other anaemias from age 2 years and in NTDT from age 10 years; the doses in the European summary of product characteristics (SmPC) are not included in the sources consulted [91]. The use of chelators in non-transfused xerocytosis with iron overload is off-label in the USA, where the indication is limited to transfusional overload and thalassaemic NTDT [88]. Chelation regimens and their detailed monitoring follow those of the thalassaemias.

7.6. Total Splenectomy

Splenectomy usually eliminates HS anaemia and reduces haemolysis markers because it removes the organ in which spherocytes are destroyed; it prolongs erythrocyte survival, not always to normal, and does not correct the membrane defect [6, 11]. In the Milan series, median Hb rose from 10.8 to 13.9 g/dL, reticulocytes fell from 337 to 51 × 10⁹/L and unconjugated bilirubin from 32.5 to 12 µmol/L; anaemia was corrected in all protein subtypes, with residual reticulocytosis and bilirubin elevation in spectrin deficiency [11, 61]. The cost is a lifelong risk of overwhelming infection with encapsulated bacteria and an increase in late vascular events (sections 7.9 and 11) [6, 11].

Indications converge across guidelines, with differences in grade and age.

In the Milan series, only 20% of patients with HS underwent splenectomy between 1980 and 2005, and the rate fell in the following decade [11]. Splenectomy in children with HS is safe in the short term: in a series of 1657 splenectomies cited by the British guideline, there were no deaths and complications occurred in fewer than 1% [6]. Reactive thrombocytosis may exceed 1000 × 10⁹/L in children [6].

The laparoscopic approach is standard for a normal or slightly enlarged spleen: less pain, shorter hospital stay, better cosmetic results and lower cost, provided it is performed by trained surgeons (BSH, grade 1, B) [6, 11]. A meta-analysis of 19 studies involving 1898 children undergoing non-traumatic splenectomy (1202 laparoscopic and 696 open procedures) showed shorter hospital stays with laparoscopy (mean difference −1.59 days; 95% CI −2.18 to −1.00) [92]. Laparoscopy is possible with a massive spleen, with longer operating time, and perioperative splenic artery embolisation helps in such cases [11]. Retained accessory spleens may cause recurrent anaemia; preoperative ultrasonography to assess spleen size is recommended, and three-dimensional computed tomography (CT) is reserved for cases requiring anatomical detail [6, 11]. Laparoscopic cholecystectomy can be performed simultaneously [6].

The French PNDS (National Diagnosis and Care Protocol) considers laparoscopy the gold standard for total splenectomy, with a normal or enlarged spleen, depending on the experience of the team, and accepts the open (midline or subcostal) and robotic approaches; embolisation or ligation of the splenic vessels as an alternative to splenectomy is not recommended outside comparative studies [8]. Regarding accessory spleens, present in about 16% of the population and capable of compensatory hypertrophy up to 3–5 cm after total splenectomy, the PNDS concludes that there are no data justifying their removal or preservation in HS and that their preservation could amount to a partial splenectomy, which diverges from the concern of the European consensus about recurrence of anaemia [8, 11]. Any splenectomy, total or partial, requires vaccination at least 15 days beforehand and postoperative antibiotic prophylaxis (section 7.10) [8].

7.7. Partial Splenectomy and Embolisation

Partial or subtotal splenectomy removes 80–90% of the enlarged spleen, aiming to reduce haemolysis while preserving immune function; it is particularly indicated below age 6 years with severe anaemia or transfusion dependence and should be performed by an experienced surgeon using an open, laparoscopic or robotic approach [11]. The British guideline considers it theoretically associated with a lower risk of sepsis but liable to require further surgery for haematological recurrence or symptomatic gallstones (grade 2, C) [6]. In the French series by Pincez and colleagues, 79 children with HS underwent open subtotal splenectomy between 1985 and 2013; among the 39 younger than 5 years (31 transfusion-dependent), followed for a mean of 12 years, transfusions fell dramatically, Hb allowed normal growth and splenic function was preserved in 96%, but haemolysis was reduced rather than suppressed, subsequent gallstones and remnant regrowth occurred, and half required total splenectomy after a median of 5 years [11]. In other series, 3 of 40 patients required total splenectomy over 1–14 years with preserved phagocytic function, 4 of 18 developed new stones, and fewer than 5% of 62 children required completion splenectomy [6]. In a series of 44 children treated between 2008 and 2020, laparoscopic total splenectomy involved less blood loss, shorter hospital stay and greater reduction in haemolysis than laparoscopic partial splenectomy, and partial splenectomy was associated with more symptomatic recurrent splenomegaly and additional operations [93]. Guizzetti's meta-analysis (14 observational studies) found a greater Hb increase with total splenectomy (3.6 versus 2.2 g/dL) and a greater reticulocyte reduction (12.5% versus 6.5%) at one year, stable results after partial splenectomy for at least 6 years, no overwhelming post-splenectomy infection (OPSI), and symptom recurrence of 5–10% after partial splenectomy, with short follow-up [11, 94]. The EHA group issued no recommendation on partial splenectomy in HS because it considered the data conflicting [11]. A Chinese series of 12 children undergoing single-port surgery with three-dimensional CT planning recommends preserving less than 10% of the upper pole to reduce recurrence [95]. Partial splenic embolisation is advocated in a 2026 Chinese narrative review as an alternative to splenectomy, without robust comparative data that would change guidelines [96]. Patients undergoing partial splenectomy or embolisation should be considered at risk of infection and follow the same preventive measures [76, 77]. GeneReviews allows antibiotic prophylaxis to be discontinued one year after partial splenectomy if the pitted red cell count demonstrates adequate splenic function [2]; counting these erythrocytes by phase contrast is the gold standard for splenic function, with an upper normal limit of 4% [77].

The French PNDS (National Diagnosis and Care Protocol) accepts offering subtotal or partial splenectomy, which usually leaves the lower pole, in severe forms (Hb <8 g/dL and/or transfusion dependence) in children under 5 years (grade B, category 2b), with possible indications, still to be validated, in children under 6 years with repeated transfusions, in the moderate form with impact on daily activity and in massive splenomegaly; it does not recommend it in adults and reserves it for centres and surgical teams experienced in the technique [8]. According to the protocol, transfusion independence is achieved in the great majority of severe childhood forms, and secondary completion, owing to regrowth of the remnant and recurrence of anaemia, is necessary in about a quarter of patients, several years later, at an age of lower infectious risk, a proportion lower than the half of severe children in the Pincez series [8, 11]. In HPP, partial or subtotal splenectomy gave a modest Hb gain with persistent transfusion requirements, and the PNDS does not recommend it in children (grade B, category 3) [8]. After partial splenectomy, the PNDS sets antibiotic prophylaxis at 1 year [8].

7.8. Cholecystectomy

Cholecystectomy is indicated for symptomatic gallstones [2, 6]. The 2004 guideline recommended always performing splenectomy in children with HS who required cholecystectomy; the 2011 update made this controversial, and the 2017 consensus recommends that indications for splenectomy in a patient undergoing cholecystectomy should not differ from those without cholecystectomy [11, 97]. In the Ruparel series, among 32 children undergoing cholecystectomy, 5 did not undergo splenectomy and none experienced biliary symptoms over a median follow-up of 15.6 years; in a series of 16 children with mild HS undergoing cholecystectomy without splenectomy, only 3 subsequently required splenectomy [6, 11]. In a child undergoing splenectomy with symptomatic stones, the gallbladder should be removed; with asymptomatic stones, cholecystectomy is controversial, and if the gallbladder is retained, including after cholecystostomy with stone extraction, close ultrasound follow-up is necessary (grade 2, C); without gallstones, the gallbladder should not be removed [6]. GeneReviews allows consideration of cholecystectomy for asymptomatic stones discovered on screening to prevent obstructive jaundice and pancreatitis [2]. In adults with mild HS, ultrasound screening for silent stones is of uncertain value; in the non-haemolytic population, only 15–20% of silent stones become symptomatic [6]. Regular ultrasonography from approximately age 5 years may identify children who will benefit from splenectomy before puberty (grade 2, C) [6].

The French PNDS (National Diagnosis and Care Protocol) is more interventionist: it recommends cholecystectomy for symptomatic gallstones (grade B, category 1), advocates it in children even with asymptomatic gallstones, given the context of chronic haemolysis (grade B, category 2B), and at the same operation as splenectomy when the gallbladder contains stones, even asymptomatic (grade B, category 1); it does not recommend it prophylactically without stones; in asymptomatic adults, it discusses it in the presence of comorbidity or residence far from care; and it prefers laparoscopy with a short stay or as a day case [8]. The approach to asymptomatic stones therefore diverges from the British guideline, which considers it controversial at the time of splenectomy [6, 8].

7.9. Thrombosis After Splenectomy

Acute splenic or portal vein thrombosis (SPVT) is an early complication: in a review by Krauth (2008), it occurred in 11 of 89 patients (12.3%) undergoing splenectomy for haemolytic disease, compared with 2 of 118 (1.7%) for immune thrombocytopenia and 0 of 122 for trauma [11]. A large spleen is a risk factor, the surgical approach does not appear to alter risk, and thrombophilia screening does not identify at-risk patients. Asymptomatic thrombosis occurs at a median of 6 days (3–11), and the European consensus recommends at least one Doppler ultrasound on day 7 after splenectomy (86% agreement; grade 2, C) [11]. Usual treatment is intravenous heparin followed by oral anticoagulation for 3–6 months; resolution was complete in 63.3%, partial in 13.3%, with persistence in 7.7% and cavernoma or portal hypertension in 15.5% [11]. The role of thromboprophylaxis is unknown; the British guideline does not indicate prolonged thromboprophylaxis after splenectomy in HS and recommends standard perioperative thromboprophylaxis in adults [6, 11].

The French PNDS (National Diagnosis and Care Protocol) estimates the risk of splenoportal thrombosis at between 5% and 51% in adults splenectomised for any indication and at no more than 5–10% in children, asymptomatic in half of cases, at a median of 6 days (3–11); it recommends a systematic Doppler between day 5 and day 8 (grade B, category 1), instead of day 7 in the European consensus, and maintains standard postoperative thromboprophylaxis in HS [8, 11]. Late events (pulmonary embolism, intracardiac thrombus, cerebral thrombosis or stroke, portal or splenic thrombosis) are rare, justify a search for associated thrombophilia and are treated without specific features [8].

Late vascular events are the other cost. In a study of HS families, the cumulative incidence of arterial events by age 70 was 22% in splenectomised women and 32% in splenectomised men, compared with 3% and 2% in affected individuals who had not undergone splenectomy; venous events were 20% and 19%, compared with 6% and 4%; the hazard ratio (HR) for splenectomised individuals was 7.2 (95% CI 2.8–17.2) for arterial events and 3.3 (95% CI 1.1–9.8) for venous events [74]. An earlier study by the same group found a 5.6-fold higher rate of atherosclerotic events in asplenic individuals older than 40 years, with the first event a decade or more after surgery [11]. Conversely, in a series of 39 adults with HS followed for a median of 25 years, there were no thrombotic manifestations [11]. Splenectomy is also a risk factor for pulmonary arterial hypertension, particularly in haemolytic diseases, and in a Danish registry the VTE risk beyond one year was approximately three times that of the general population after splenectomy for trauma and higher in haemolytic anaemias [11]. The British guideline calls for these risks to be weighed in the decision [6].

The PNDS considers an echocardiogram with estimation of pulmonary pressures every 3 to 5 years reasonable in splenectomised patients and in the presence of any clinical sign, with referral to a pulmonary hypertension reference centre if there are signs of pulmonary arterial hypertension; and it cites registry studies that suggest an excess of solid and haematological cancers after splenectomy, greater when the indication is not traumatic [8].

In stomatocytoses, splenectomy is contraindicated (EHA, 100% agreement; grade 2, C; BSH, avoid in some forms, grade 1, B) [6, 11]. In 1996, Stewart and colleagues described nine splenectomised adults from seven families with severe recurrent late thrombotic complications (deep vein thrombosis, pulmonary embolism, portal thrombosis, intracardiac thrombus, arterial thrombosis, pulmonary hypertension), four of whom died, with no complications in six affected individuals who had not undergone splenectomy; splenectomy was only partially effective in OHSt and ineffective in DHSt [11, 98]. In the French series, 75% of splenectomised patients with xerocytosis developed thrombosis, including all eight with PIEZO1, with prolonged anticoagulation; the four splenectomised patients with KCNN4 had no thrombosis over a mean follow-up of 26.5 years but continued to have haemolysis [18, 38]. Coinheritance of sickle cell trait or β-thalassaemia trait with α-triplication was associated with severe thrombosis in that series [18]. In patients with stomatocytosis diagnosed as HS who have already undergone splenectomy, the European consensus suggests considering lifelong anticoagulation [11].

In DHSt, the French PNDS (National Diagnosis and Care Protocol) recommends long-term therapeutic anticoagulation after a thrombotic event in a splenectomised patient, in the absence of contraindication, because of the high risk of recurrence; in splenectomised patients without thrombosis there are no data on primary prevention with an anticoagulant or antiplatelet agent, and the decision rests with a multidisciplinary meeting (vascular medicine, haematology, internal medicine, cardiology) according to cardiovascular risk factors, family history and thrombophilia testing, with strict control of risk factors and venous thrombosis prevention measures for all; non-splenectomised patients do not need prophylaxis [8]. Only one splenectomised patient with KCNN4 with thrombotic complications (stroke and venous thrombosis) had been described at the time of the protocol [8].

7.10. Prevention and Treatment of Infection in Asplenia

OPSI risk is greatest with encapsulated bacteria (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae); asplenia is also a risk factor for severe malaria, Capnocytophaga infection after animal bites, babesiosis after tick bites and Bordetella holmesii infection [11, 76]. Risk varies with the indication for splenectomy (intermediate in HS, higher in other hereditary anaemias and malignancies), age (highest before age 5 and above 60) and time since surgery (highest during the first 1–3 years), but persists throughout life, with cases decades later [11, 77]. Mortality from encapsulated bacterial sepsis after splenectomy was 50–70% in the literature reviewed by the Canadian Paediatric Society (CPS), highest below age 2 years [76]. In a Finnish cohort of 141 splenectomised children, 10 of the 11 patients with sepsis had an additional immunodeficiency, and in a German cohort of severe sepsis, pneumococcal aetiology accounted for 42% in splenectomised patients versus 12% in those with a functional spleen, with fewer than half of OPSI cases previously vaccinated [11].

In the French PNDS (National Diagnosis and Care Protocol), the pneumococcus causes more than half of severe post-splenectomy infections; the risk is highest in the first year, in the first 2 years and before 5–6 years of age, which usually contraindicates total splenectomy before that age, and persists lifelong; the recommendations are systematic antibiotic prophylaxis (grade A, category 2A), vaccination before splenectomy against pneumococcus, meningococci and Haemophilus influenzae (grade A, category 2A) and pre-emptive antibiotic active against the pneumococcus for fever >38.5 °C (grade B, category 2A) [8].

Measures rest on three pillars: patient education to act immediately with fever, vaccination and antibiotic prophylaxis [11]. The 2024 British guideline on asplenia and hyposplenism, replacing those of 1996, 2002 and 2011, recommends written or electronic information and an alert card (grade 1A), flagged clinical records with vaccination status (1A), advice on travel, malaria and bites (1B), and ideally local and national registries of at-risk patients (2C) [77]. Adherence is low: in an Italian study of 1579 splenectomised patients between 2015 and 2020, only 27–31% had received each recommended vaccine, and in France only 24% had received all four; the Australian Spleen Australia registry was associated with a 69% reduction in infection risk among registered patients [77]. Many patients splenectomised for HS cease hospital follow-up, and the general practitioner should take responsibility for vaccination and follow-up; it is also important to identify adult relatives splenectomised in the past who may be inadequately protected [6, 77].

Vaccination

Vaccines should ideally be given at least 2 weeks before an elective splenectomy or 2 weeks after (1B), but may be given at any time (2B); in one study, the functional response was better with vaccination 14 days after surgery than immediately after [77]. The GeneReviews recommendations (23-valent pneumococcal polysaccharide, PPSV23, and 13-valent conjugate vaccine, according to the Advisory Committee on Immunization Practices, ACIP, of 2010 and 2013) are outdated [2]. The vaccines concerned are the pneumococcal conjugate (PCV) and polysaccharide vaccines, the meningococcal conjugate vaccines against serogroups A, C, W and Y (MenACWY) and against serogroup B (MenB), and the Haemophilus influenzae type b (Hib) vaccine. The current schedules of the US Centers for Disease Control and Prevention (CDC) and of the United Kingdom diverge, and the British guideline itself asks each country to adapt the recommendations to its schedule [77]. The French PNDS (National Diagnosis and Care Protocol) requires the first doses at least 15 days before surgery, or 15 days after in an emergency splenectomy, and follows the 2021 French vaccination schedule [8].

Sources: [8, 77, 99–102].

The legal status of the US schedule is unstable: by a preliminary order of 16 March 2026, in the case American Academy of Pediatrics et al. v. Kennedy et al. (US District Court for the District of Massachusetts), the ACIP votes of June, September and December 2025 and a January 2026 memorandum from the acting CDC director were stayed, and the 2 July 2025 schedule became the schedule in force [102]. PCV21 does not contain serotype 4, relevant in regions of the western USA where this serotype causes ≥30% of invasive pneumococcal disease (IPD) [100]. In the United Kingdom, PPV23 remains the basis of individual protection, because it covers 11 additional serotypes, and repeated PPV23 doses less than 5 years apart may blunt the response [77]. Live vaccines can be given safely to asplenic patients [77]. In Portugal, Norma no. 011/2015 of the Direção-Geral da Saúde, updated on 1 November 2021, provides for adults in groups at increased risk of invasive pneumococcal disease Pn13 followed by Pn23 at least 8 weeks later (ideally 6 to 12 months) and a single Pn23 booster after 5 years [103]. The sources consulted do not include the Portuguese paediatric recommendations for asplenia or those of the Brazilian National Immunisation Programme. The French PNDS (National Diagnosis and Care Protocol) schedule predates the higher-valency conjugate vaccines (PCV15, PCV20, PCV21) included in later schedules, and its five-yearly meningococcal boosters bring it closer to the CDC and further from the British guideline [8, 77, 101]. In France, the PCV13 followed by PPSV23 schedule has been replaced: since 2023, at-risk adults receive a single dose of a high-valency conjugate vaccine [104, 105], and the HAS recommendation validated on 23 July 2026 provides for PCV20 in infants (3+1 schedule) and as a single dose in at-risk children and adolescents aged 15 months to 18 years, abandoning PCV13, PCV15 and PPSV23; in at-risk adults aged 18 to 64 years and in all adults aged 65 years or more, PCV21 is preferred, as a single dose [106].

Antibiotic Prophylaxis

Prophylaxis duration is the greatest point of divergence between societies because of the lack of trials: the main evidence comes from sickle cell disease, in which penicillin reduced infections by 84% in a controlled trial [76, 77]. The 2011 British HS guideline proposed offering lifelong prophylaxis to everyone, particularly below age 16 and during the first 2 years after splenectomy [6, 76]. The 2024 British guideline recommends prophylaxis for everyone after splenectomy, immediately postoperatively and for 1–3 years, and for all asplenic children until age 5 years with at least 2 full years; lifelong prophylaxis for patients at continued high risk (younger than 5 years, older than 65 years, previous IPD, haematological malignancy with immunosuppression) (1B); others may discontinue after 1–3 years with counselling (2C) [77]. The CPS recommends a minimum of 2 years after splenectomy and all children until age 5 years, and considers lifelong prophylaxis ideally recommended, weighing adherence, access, resistance and previous sepsis [76]. The AAP Red Book, cited by the CPS, proposes prophylaxis until age 5 years and for at least 1 year after splenectomy in older children with complete pneumococcal vaccination, and Spleen Australia a minimum of 3 years [76, 77]. GeneReviews notes the controversy between 3 years and lifelong treatment [2].

In the first 3 months, the combination with clavulanic acid also covers Escherichia coli and Klebsiella [76]. The CPS does not give the erythromycin dose and the 2024 British guideline gives no doses; the French PNDS (National Diagnosis and Care Protocol) gives them (following table) [8, 76, 77]. Oral penicillins are the choice in areas of low pneumococcal resistance, and the final choice should follow the local microbiology protocol [77].

Source: PNDS 2021, appendix 8 [8]. The PNDS erythromycin doses are total daily doses (250 mg/day from 2 to 8 years and 500 mg/day above 8 years and in adults) [8], whereas the 2026 Nottinghamshire protocol, which refers to the BNF and BNFc, gives 125 mg twice daily from 1 to 23 months, 250 mg twice daily from 2 to 7 years and 500 mg twice daily from 8 years and in adults, that is, double the French daily dose; in the same situation, azithromycin remains a fallback alternative (250 mg once daily) [107]. The PNDS acknowledges the lack of consensus on duration and on prophylaxis in true penicillin allergy, and extends it after pneumococcal sepsis or in particular immunosuppression [8]. The French duration (2–5 years in children, 1–2 years in adults) lies between that of the 2024 British guideline (1–3 years and up to 5 years of age, lifelong at high risk) and the minimum of the CPS (2 years), and the paediatric penicillin V dose is calculated by weight, whereas the CPS uses fixed doses by age [8, 76, 77].

Fever and Emergency Antibiotics

All patients should have a rescue antibiotic for emergency use (1A), and any infection despite preventive measures requires parenteral antibiotics and urgent hospital admission (1A) [77]. Fever requires immediate medical assessment, and blood culture should not delay antibiotics: ceftriaxone 100 mg/kg/dose, maximum 2 g per dose; where pneumococci with intermediate or high resistance are prevalent, add vancomycin 60 mg/kg/day divided every 6 hours; in severe allergy to penicillin and cephalosporins, vancomycin and ciprofloxacin, without a dose specified in the source [76]. GeneReviews specifies ceftriaxone at the meningitis dose, 100 mg/kg/day, up to 2 g/day, as a single daily dose [2]. In a patient receiving prophylaxis, fever should be treated with a different antibiotic class from that used for prophylaxis [77]. For travel to malaria-endemic areas, chemoprophylaxis, mosquito nets and repellent are advised [76, 77].

The French PNDS (National Diagnosis and Care Protocol) and its care and emergency card call for urgent consultation for fever >38.5 °C and, if rapid consultation is not possible, pre-emptive antibiotic at home: in children, amoxicillin (or amoxicillin with clavulanic acid) 45 mg/kg as the first dose and then 80 mg/kg/day in 2–3 doses, with no weight-based maximum given in the source, with urgent consultation; in adults, 2 g as the first dose and then 3 g/day in 2–3 doses; a macrolide in allergy; and, with signs of severe infection (malaise, purpura, deterioration in general condition, drowsiness), a 3rd-generation cephalosporin intravenously or intramuscularly without delay [8]. The French card records prophylaxis, vaccination dates and risk situations (bites, travel to tropical or malarial areas, which justify a prior travel medicine consultation), and the patient should always carry an in-date pack of the antibiotic, including when travelling; the family should identify other splenectomised members and check their vaccination [8].

7.11. Haematopoietic Stem Cell Transplantation

Allogeneic HSCT is curative because it replaces erythropoiesis but is reserved for severe recessive forms that remain transfusion-dependent despite splenectomy. In 2026, Koçak Göl and colleagues described an adolescent girl with homozygous SPTA1-related HS, dependent on transfusions every 2 or 3 weeks after splenectomy and with liver fibrosis from iron overload, successfully transplanted from a human leucocyte antigen (HLA)-identical sibling (section 10) [12]. The authors cite previous transplants in HS, including a patient with another homozygous SPTA1 variant, and advocate including HSCT in algorithms for refractory forms; in the published reply, Doğru and colleagues highlight transplant-related mortality, chronic graft-versus-host disease (GVHD), infertility, growth retardation and secondary malignancies, which should be weighed against the life expectancy afforded by modern chelation and transfusion support; HSCT should remain exceptional, considered early in patients with severe biallelic SPTA1 disease [12]. In the Chonat series, patients with almost complete α-spectrin deficiency required lifelong transfusions with chelation or transplantation [3, 42].

7.12. Management of Other Membrane Disorders

In HE, most patients need no treatment, only full blood count and reticulocytes; symptomatic patients receive folate, transfusion in severe or aplastic episodes and surveillance for gallstones, and splenectomy is kept for severe anaemia or transfusion dependence, after vaccination and with shared decision-making [3]. No guideline specifically addresses elliptocytosis; the British guideline is an HS guideline [3]. HPP often requires transfusions in the first year and splenectomy later in severe forms [3, 5]. Isolated SAO needs no treatment [3]. The French PNDS (National Diagnosis and Care Protocol) treats HPP like moderate to severe HS with regard to neonatal jaundice, acute anaemia, folate, splenectomy (Hb <8 g/dL and/or transfusion dependence, grade B, category 1; after 5 years if possible; laparoscopic approach) and cholecystectomy; erythropoietin may be used in the newborn by analogy with HS, without data in HPP; and simple HE is not exposed to the complications of chronic haemolysis and needs no haematological follow-up [8].

In xerocytosis, treatment is supportive: transfusion when needed and surveillance and treatment of iron overload, by phlebotomy in patients with preserved Hb or by chelation [18, 70]. The Frederiksen review gathered 73 articles published between 1971 and 2019, all but one case reports or case series, with no randomised trial or society guideline, and recommends international registries [70]. Folate and surveillance of Hb, reticulocytes, gallstones and iron also apply to OHSt [3]. Splenectomy is contraindicated in all stomatocytoses (section 7.9) [11, 72]. In Rh deficiency syndrome, early recognition, molecular characterisation, enrolment in a rare donor programme, autologous donation and typing of relatives are the main measures [3]. In pseudohyperkalaemia, K+ should be measured on a separate sample immediately to avoid inappropriate treatment [3, 5]. SLC4A1-related dRTA with anaemia is treated with oral alkali, transfusion and iron as needed [23].

The French PNDS (National Diagnosis and Care Protocol) describes the management of DHSt in more detail [8]. Iron overload is usual in adults, more frequent with PIEZO1, and should be sought systematically in adults, with liver MRI combined with ferritin and transferrin saturation, and in children and adolescents with ferritin persistently >500 µg/L; liver biopsy is not first line and is discussed only with a hepatologist in case of doubt [8]. In non-anaemic patients, phlebotomies, usually well tolerated, are the choice and reduce the manifestations of hyperviscosity in polycythaemic patients, with monitoring by ferritin, Hb and tolerance, follow-up MRI after depletion and maintenance phlebotomies; in anaemic patients or those with poor tolerance, chelators (deferasirox, desferrioxamine) are used, with good efficacy on the tissue load and outside the marketing authorisation in France [8]. Depletion treatment starts in the presence of tissue overload on MRI, with a realistic threshold of 100 µmol/g (5–7 mg/g), modulated by age, with no threshold validated in the literature and no randomised trials on the modalities [8]. The PNDS does not set doses for phlebotomy or chelators; the doses of deferasirox are in section 7.5, and their application to non-transfused DHSt is an extrapolation from thalassaemic NTDT [88]. There is no specific transfusion threshold, systematic phlebotomy in polycythaemia is not recommended outside iron overload or a clinical hyperviscosity syndrome, and there are no data on systematic antiplatelet therapy [8]. Hepatitis B vaccination is offered because of the possibility of transfusion [8]. Pseudohyperkalaemia should not lead to prescription of potassium-lowering treatment without confirmation on a sample kept at 37 °C or analysed immediately, and splenectomy is discussed only in exceptional situations in a specialised centre, including repeated painful splenic infarctions, which may require step III analgesics [8].

7.13. Regulatory Status

Sources: [3, 85, 88–91, 108, 109]. The dates of initial FDA authorisation of mitapivat and of the European extension to thalassaemia are not included in the sources consulted.

7.14. Trials and Comparative Studies

Sources: [92–94, 110–114].

None of these studies is randomised. SATISFY is a single-arm trial without a comparator, with results known only through a conference abstract and an oral presentation whose transcript is of poor quality; publication of results from the 32-week core period, announced by third parties for 2026, was not found in Europe PMC or Crossref on 10 October 2026 [111, 112]. The Hb response of 57–61% refers to a secondary outcome in a small, mixed, uncontrolled group and cannot be compared with mitapivat trials in other diseases. Surgical meta-analyses compare observational series with different indications and follow-up: partial splenectomy is chosen for younger and more severely affected children, biasing the comparison; the absolute Hb increase should be read alongside the reintervention rate (approximately half of severely affected children in the Pincez series), rather than as intrinsic superiority of one technique [11, 94]. The phase 3 senicapoc trial in sickle cell disease improved haemolysis markers without reducing vaso-occlusive crises, the only notable negative result in this field [3, 114].

7.15. Developments in the Last 12 Months

Also ongoing are a French genotype–phenotype correlation study in xerocytosis (NCT06892171, 20 patients, expected completion in 2028), an Egyptian study of full blood count parameters in paediatric HS (NCT07842718) and the European rare anaemia epidemiological platform RADeep (NCT06213402) [117]. The absence of other targeted therapies in clinical development in the searches performed does not prove that none exist.

7.16. Agents and Interventions to Avoid

Splenectomy should be avoided in all stomatocytoses and in Gardos channelopathy, and no splenectomy should be performed without a firm diagnosis [3, 11]. Iron preparations should be given only for documented iron deficiency [2]. GeneReviews advises against contact sports in the presence of splenomegaly, although the British guideline finds no evidence to restrict activity in children with HS [2, 6]. Routine concomitant splenectomy during cholecystectomy is no longer recommended [11]. Antipneumococcal antibody measurements have no role, and repeated PPV23 doses less than 5 years apart may attenuate the response [77]. Use of mitapivat or senicapoc outside a clinical trial has no basis in the sources consulted.

7.17. Pregnancy and Lactation

In HS, pregnancy requires folic acid (800–1000 µg daily in GeneReviews) and monitoring of the full blood count and reticulocytes, because anaemia may worsen to the point of requiring transfusion, especially in non-splenectomised women [2, 6]. The AGLT is falsely positive in about one third of pregnant women, which limits a new diagnosis during pregnancy [5]. Prospective parents should be informed of the risk of neonatal jaundice, and the newborn should be monitored for several days [6]. In pregnancies with a fetus at risk of severe recessive HS, MCA Doppler surveillance allows detection of fetal anaemia and planning of intrauterine transfusions [12, 64]. When a parent has xerocytosis, ultrasound at least monthly is recommended, whatever the gene [18, 73]. In DHSt, the French PNDS (National Diagnosis and Care Protocol) recommends monitoring any pregnancy of a couple with an affected parent (50% transmission risk) in a level 3 maternity unit, with monthly obstetric ultrasound from the 3rd month and more often if an effusion appears; severity ranges from increased nuchal translucency to pleural and pericardial effusions, chylous ascites, hygroma and anasarca; the decision on drainage taps in utero is taken at a multidisciplinary meeting; fetal anaemia, inconsistently associated with anasarca, occurred in up to 18% in a recent series; and folic acid is given from the preconception period and throughout pregnancy, even with compensated haemolysis [8]. In Rh deficiency syndrome, anti-Rh29 alloimmunisation exposes to haemolytic disease of the fetus and newborn [3]. Iron chelators, erythropoietin and mitapivat have not been evaluated in pregnancy in the sources consulted, and the sources do not address breastfeeding.

7.18. Perioperative Care

Before splenectomy, the diagnosis must be confirmed (excluding stomatocytosis and CDA II), vaccinations completed at least 2 weeks beforehand, and the gallbladder and spleen size assessed by ultrasonography; afterwards, perform splenoportal Doppler ultrasonography on day 7, start antibiotic prophylaxis, provide the alert card and emergency antibiotic, and use standard perioperative thromboprophylaxis in adults [6, 11, 77]. Vaccines not given beforehand should be administered 2 weeks afterwards, ensuring the patient is not lost to follow-up after discharge [77].

7.19. Transition to Adulthood

Transition is the point at which prophylaxis, vaccine boosters and gallstone and iron surveillance are lost, because many splenectomised patients cease hospital follow-up [77]. Young people should know their diagnosis, the gene when known, the risk of severe infection and what to do with fever, the risk of thrombosis and the reproductive risk; coinheritance of haemochromatosis alleles and increased iron absorption justify measuring ferritin in adults [6]. Adults with mild disease usually do not require regular follow-up except for these issues [6].

The French PNDS (National Diagnosis and Care Protocol), without specific recommendations for the membrane disorders, proposes repeating the disclosure of the diagnosis in early adolescence, assessing the knowledge and beliefs of the patient and family, developing autonomy in emergencies (fever in the splenectomised patient, worsening anaemia, abdominal pain, jaundice) and in daily management (medication, prescriptions, appointments), identifying future contacts and the pathways for consultation, admission and emergency care, explaining the differences between paediatric and adult teams and ensuring a general practitioner as a fixed point; the French inter-network transition group for rare diseases provides online tools [8]. In education, the protocol highlights the signs of worsening haemolysis (jaundice, dark urine, pallor, unusual tiredness, feeding difficulty), which call for a full blood count with reticulocytes and possibly parvovirus B19 serology, postprandial right upper quadrant pain, which calls for ultrasound, attendance at appointments even without symptoms and exemption from an individualised school care plan, unless anaemia is very symptomatic [8].

7.20. Guidelines and Differences

Sources: [2, 5, 6, 8, 11, 75–77, 97, 99–102].

Sources: [2, 5, 6, 8, 11, 76, 77, 85, 86, 101, 106].

8. Reproductive Options and Family Counselling

8.1. Recurrence Risks by Mode of Inheritance

Counselling depends on the mode of inheritance, which differs between genes and, for the same gene, between families; identifying the gene therefore has greater consequences for counselling than for management [3, 5]. In dominant HS (ANK1, SPTB, SLC4A1), each child of an affected parent has a 50% risk of inheriting the variant; severity tends to be similar within the family but may vary through coinheritance of low-expression alleles or modifiers [5, 6]. In recessive HS (SPTA1, EPB42 and recessive forms of ANK1, SLC4A1 and SPTB), when both parents are heterozygotes, each sibling of the proband has a 25% risk of being affected, a 50% risk of being an asymptomatic carrier and a 25% risk of not being a carrier; the proband's children are obligate heterozygotes, and each sibling of the parents has a 50% risk of being a carrier [2]. In EPB42-related HS, heterozygotes are asymptomatic and are not at risk of developing the disease [2]. In families with HE and HPP, at least one parent usually has HE; two parents with HE, or one parent with HE and another carrying αLELY, may have a child with HPP [5, 31]. In SAO, each child of a carrier has a 50% risk of inheriting it; when both parents transmit SLC4A1 variants, the risk of recessive dRTA should be discussed [3, 5]. Hereditary stomatocytoses are dominant, with a 50% transmission risk, and Rh deficiency syndrome is recessive [3, 5].

8.2. De Novo Variants, Mosaicism and Apparent Recessivity

De novo variants are not rare and explain many cases of “recessive” ANK1- and SPTB-related HS that are subsequently transmitted dominantly [5, 6]. For a child with severe HS and clinically normal parents, the genetic differential diagnosis includes recessive inheritance (SPTA1, αLEPRA in trans), a dominant de novo variant and reduced penetrance in a parent; recurrence risk is 25% in the first scenario and low in the second, but not zero because of possible germline mosaicism [2, 5]. In EPB42-related HS, when only one parent is a carrier, with confirmed paternity and maternity, the second variant may be de novo in the proband, postzygotic in a mosaic parent, or uniparental isodisomy may be present [2]. In Gardos channelopathy, the recurrent variant p.(Arg352His) also occurs de novo, and expressivity ranges from severe anaemia to no anaemia in carrier parents [3, 38]. Molecular testing of parents and all relatives is therefore useful when the proband's phenotype is more severe than that of relatives [3].

8.3. Cascade Testing and Carriers

Asymptomatic older and younger siblings should be assessed with a full blood count, reticulocytes, blood film and osmotic fragility or ektacytometry, and testing for familial variants when known [2]. Carrier testing in recessive forms requires prior identification of familial variants [2]. Adult relatives splenectomised in the past should be identified to update vaccination and prophylaxis [6]. In families with xerocytosis, family investigation identifies patients with compensated haemolysis and previously undiagnosed iron overload [18]. In Rh deficiency syndrome, relatives should be typed as potential compatible donors [3].

In DHSt, the French PNDS (National Diagnosis and Care Protocol) recalls the patient's legal obligation to inform relatives (decree no. 2013-527 of 20 June 2013), offers relatives a full blood count, a complete haemolysis work-up and genetic testing, because they may be asymptomatic or polycythaemic, and asks that the risk of perinatal oedema be assessed and iron overload sought in diagnosed relatives [8]. In simple HE, family screening is offered to first-degree relatives, and the feature should be recalled when the carrier wishes to have children, to assess the risk of a more symptomatic form in the couple's offspring [8].

8.4. Pregnancy with Known Risk

The ideal time to discuss risk and options for prenatal diagnosis and preimplantation genetic testing is before pregnancy; counselling should be offered to young adults who are affected, carriers or at risk [2]. Prenatal diagnosis and preimplantation testing are possible once familial variants have been identified, and views on their use differ between professionals and families [2]. They carry greater weight in severe recessive forms, with a risk of hydrops and transfusion dependence [42, 64]. In pregnancies at risk of severe HS, MCA Doppler surveillance and planning delivery at a centre with paediatric haematology and the capacity for intrauterine transfusion are the measures described [12, 64]. When a parent has xerocytosis, ultrasonography at least monthly targets perinatal oedema, and when a parent has Gardos channelopathy, the risk of hydrops, although described in only one family, justifies the same surveillance [18, 73]. The newborn of a parent with HS should undergo bilirubin surveillance during the first week and Hb monitoring during the first 2–4 months; in families with SPTB variants, bilirubin surveillance is justified even with a normal blood count [2, 3]. Folate may be considered in infants with chronic haemolytic anaemia and significant reticulocytosis [2].

8.5. Counselling of Minors and Consultation Content

Assessment of minor siblings with a full blood count and membrane tests is justified by direct clinical benefit, because it enables prevention of kernicterus in newborns and recognition of an aplastic crisis or gallstones [2, 6]. In recessive forms, carrier status in a healthy minor does not alter haematological surveillance, and GeneReviews directs reproductive discussion towards young adults who are affected, carriers or at risk [2]. The consultation should cover the mode of inheritance and recurrence risk, variability in severity, warning signs (neonatal jaundice, aplastic crisis, fever after splenectomy), the meaning of a VUS and the reason not to perform splenectomy without a firm diagnosis [2, 5, 11].

The French PNDS (National Diagnosis and Care Protocol) calls for disclosure of the diagnosis in a dedicated consultation with a physician knowledgeable about the membrane disorders, if possible with both parents, explaining chronicity, complications, signs of poor tolerance of anaemia, the indications for splenectomy and the mode of inheritance, with construction of the pedigree and identification of relatives who may benefit from screening [8]. When one member of a couple has HS, it recommends screening the partner for constitutional haemolysis, because co-inheritance may give more severe forms in the children; and, when a couple is referred directly for genetic counselling, for example in early pregnancy, the geneticist should obtain documents confirming the diagnosis made by a specialist, or first refer the couple to that specialist [8].

9. Special Considerations: Population Diversity and Epidemiology

9.1. Prevalence and What Each Figure Measures

The HS figures do not measure the same thing. The estimate of 1:2000 is a review estimate for Northern European and North American populations [5, 7]. The 1.1% figure in German blood donors measures an abnormal result on a modified glycerol lysis test, in a population of healthy people, and indicates that very mild forms or the carrier state are frequent, not that 1% of the population has clinical disease [118]. The US birth prevalence comes from a 1962 study, with the methods of the time [1, 28]. HS is much less frequent outside populations of Northern European ancestry [3]. For DHSt, the Orphanet records include classes and values that are inconsistent with one another (worldwide <1/1,000,000; North America 0.1/100,000 with class 1–9/100,000; Denmark 0.7/100,000 with class 1–5/10,000), and the difference between 1:10,000 and 1:50,000 in the two main sources reflects different methods and probable underdiagnosis [5, 11, 17]. The true incidence of HE is uncertain, because most heterozygotes are asymptomatic and never reach diagnosis [3]. The prevalence of SAO differs between the ICSH (1–1.5%) and the French PNDS (National Diagnosis and Care Protocol) (5–20%), because the higher values refer to coastal regions of endemic malaria [5, 8].

9.2. Gene Distribution and Founder Effects

Percentages by gene depend on the population, the method (protein or DNA) and patient selection, and should not be transposed from one population to another. In the Chinese series, variants were identified in 79 of the 94 patients studied (84%) [59]. Wang and colleagues found SPTB to be the most frequent gene in eastern China, unlike the European series, and the EPB42 variant p.Gln377His (NM_000119 numbering; = NM_001114134.2:c.1041G>T p.(Gln347His)) to be a hotspot [59]. In Japan, the 4.2 Nippon and 4.2 Fukuoka variants are founder variants, but ancestry is a clue and not a filter, because 4.2 Nippon has been described in an Italian patient [2, 3]. The SPTB variants Providence, Buffalo and Chiang Mai are common in Thailand [44]. SAO and the spectrin variants of HE are concentrated in regions of endemic malaria, through selection, and SLC4A1-related recessive dRTA occurs mainly from the Middle East to Melanesia [5, 23, 26]. The αLELY allele is ancient and stable, with frequencies of 20–31% in all groups studied [31]. According to the French PNDS (National Diagnosis and Care Protocol), de novo HS variants are concentrated in ANK1 and SPTB, and about 25% of patients have no family history [8].

9.3. Data from Portugal and Brazil

The most precise Portuguese finding in the sources consulted concerns the 4.2 Lisboa allele: in 1995, Hayette and colleagues described a 26-year-old Portuguese woman with recessively inherited hereditary haemolytic anaemia and absent protein 4.2, homozygous for a single-nucleotide deletion in exon 2 of EPB42 creating a premature stop codon; her parents and one brother were heterozygotes, and there were no manifestations other than anaemia [58]. Ankyrin Florianópolis, named after the Brazilian city, was described in three unrelated probands of different genetic backgrounds with severe dominant HS; the sources consulted do not describe these patients' geographical origin [6]. In South America, the sources consulted include an Argentinian study of EMA performance in 181 samples and an Argentinian series of 20 children with DHSt [79, 84]. The sources consulted do not provide prevalence estimates for HS or other membrane disorders in Portugal or Brazil, which does not demonstrate the absence of published data.

9.4. Special Populations

In newborns, HS is an important cause of severe hyperbilirubinaemia and kernicterus, and diagnosis is more difficult because the blood film is atypical and OF unreliable [6, 65]. In pregnant women, anaemia worsens and AGLT yields false positives [5]. In older people, HS may first be diagnosed after an infection [5]. Athletes with DHSt may have exercise-induced haemolysis [5]. Patients with the Rh-null phenotype present a transfusion medicine and rare donor challenge [3]. Asplenic travellers to malaria-endemic areas are at risk of severe malaria [11, 76]. In SAO carriers, coinheritance with α-thalassaemia does not worsen the presentation, but coinheritance with HbE, α+-thalassaemia or HbH disease has been described in compound heterozygotes with dRTA [5].

9.5. Reference Networks and Patient Associations

ERN-EuroBloodNet is the European Reference Network for Rare Haematological Diseases, one of the 24 European networks; it offers the Clinical Patient Management System (CPMS) for virtual case consultation between specialists, the ENROL registry, guidelines and clinical decision tools, online training in rare anaemias and erythrocyte morphology, and patient representatives (ePAG) [13]. The network promotes the SATISFY trial and the RADeep rare anaemia platform [110, 117]. Its patient organisations page lists eleven pan-European networks (for example, the Thalassaemia International Federation and the European federation of haemochromatosis associations) and does not include an organisation specifically for spherocytosis or red cell membrane disorders [13]. GeneReviews refers readers to the MedlinePlus “Hereditary spherocytosis” page [2].

In France, red cell membrane disorders belong to the MCGRE rare disease health network (filière de santé maladies rares; constitutional rare disorders of the red cell and erythropoiesis), which maintains online the list of reference and competence centres, with the disorders followed in each; the PNDS (National Diagnosis and Care Protocol) was coordinated by the reference centre for major sickle cell syndromes, thalassaemias and other rare disorders of the red cell and erythropoiesis at Hôpital Bicêtre (AP-HP) [8]. Severe forms with chronic anaemia, or with iron overload or thrombotic complications, are admitted to ALD no. 10 (severe constitutional chronic haemolyses), with a care protocol shared between the specialist and the general practitioner [8]. Diagnosis and initial work-up involve paediatricians, neonatologists, haematologists, internists, emergency physicians, laboratory scientists, geneticists and transfusion physicians, joined by anaesthetists, surgeons, obstetricians, midwives and radiologists, and coordination rests with the paediatrician or the physician specialised in constitutional red cell disorders [8]. Molecular diagnosis is performed in accredited laboratories with panels declared to the Agence de la biomédecine; the patient association Vigifavisme took part in the review of the protocol, and the inter-network transition group for rare diseases provides tools for the move to adult medicine [8].

10. Illustrative Clinical Case

In 2026, Koçak Göl and colleagues published in the Turkish Journal of Hematology the case of a 14-year-old girl with severe recessive HS (full text at https://pmc.ncbi.nlm.nih.gov/articles/PMC13225704/) [12]. Anaemia began in utero and required intrauterine transfusions. After birth, the patient required two plasma exchange procedures and intensive phototherapy for refractory anaemia and indirect hyperbilirubinaemia, and during childhood she had hepatosplenomegaly and repeated haemolytic crises. In the neonatal period there was reticulocytosis, normoblasts on the blood film and a positive DAT, a finding not characteristic of HS and unexplained in the report; OF and EMA were not diagnostic, which the authors attributed to early transfusions masking the patient's erythrocyte profile. Elevated neonatal transaminases (aspartate aminotransferase 502 U/L, alanine aminotransferase 302 U/L) prompted suspicion of neonatal hepatitis or primary haemochromatosis, treated with desferrioxamine, N-acetylcysteine, vitamin E and selenium. Haemoglobin studies (HbF below 0.5, HbA2 2.1 and HbA 97.4, without units in the report) and testing for the variants Cd2 T>C and IVS II-16 G>C did not explain the severity [12].

The subsequent course brought complications of treated disease: mild left ventricular non-compaction and grade IV transfusional hepatic iron overload with focal perisinusoidal and periportal fibrosis on biopsy. Despite splenectomy and cholecystectomy, the patient remained dependent on transfusions every 2 or 3 weeks [12]. Clinical exome sequencing identified homozygous SPTA1 c.7134G>A, absent from gnomAD, with a splicing effect predicted by SpliceAI and dbscSNV, heterozygous parents, and classified as likely pathogenic by ACMG/AMP criteria, establishing HS type 3 [12]. The publication does not specify the transcript; in NM_003126.4, the variant corresponds to p.(Gln2378=), a synonymous substitution with a predicted splicing effect, and is not recorded in ClinVar [45]. According to the caption, figure 1 of the article shows the homozygous variant in the Integrative Genomics Viewer (IGV) [12].

Because of iron overload and liver fibrosis, the patient underwent allogeneic HSCT from an HLA-identical sibling, with “Protocol 26” conditioning (busulfan, cyclophosphamide and antithymocyte globulin), chosen to limit hepatic toxicity, together with antifungal prophylaxis and rigorous infection control. There was no acute or chronic GVHD, severe infection or organ injury; in the fourth year after transplantation, the patient is transfusion-independent with full chimerism [12].

The case brings together several themes of this article: fetal presentation of recessive SPTA1 forms, failure of membrane tests in a patient transfused since birth, late genetic diagnosis, transfusion dependence persisting after splenectomy when residual α-spectrin is almost absent, as in the Chonat series, and iron overload with liver injury [12, 42]. The authors' conclusion, proposing inclusion of HSCT in algorithms for refractory forms, goes beyond the guidelines: none of the guidelines consulted includes HSCT, and the reply by Doğru and colleagues argues that transplantation should remain exceptional, weighed against mortality and late morbidity [6, 11, 12]. The measures described during the neonatal period (plasma exchange, chelation and antioxidants for suspected haemochromatosis) are management decisions in this case, not routine care.

A second case illustrates the reverse error, a membrane disorder mistaken for another disease. In 2025, Molina-Arrebola and Bain described a man referred in 1997 for suspected hereditary haemochromatosis, with Hb 166 g/L, MCV 112.7 fL, transferrin saturation 90.7%, ferritin 956.5 ng/mL, total bilirubin 1.82 mg/dL and grade IV hepatic haemosiderosis with septal fibrosis on biopsy; he was treated for years with phlebotomy for haemochromatosis with Gilbert syndrome despite negative HFE testing [71]. Blood film review showed stomatocytes, knizocytes, irregularly contracted cells, spherocytes, target cells and occasional schistocytes, with reticulocytes persistently near 230 × 10⁹/L, OF showing increased resistance and a marked increase in hyperchromic cells on the cytogram; the haemolytic anaemia panel identified PIEZO1 c.5975C>T p.(Thr1992Met) in apparent homozygosity, classified as a VUS in ClinVar and considered pathogenic by the authors [53, 71]. The patient continues phlebotomy, and liver ultrasonography has normalised. Iron overload in xerocytosis may be disproportionate to anaemia, and diagnosis matters both for iron management and for avoiding splenectomy [71].

11. Prognosis and Long-Term Follow-Up

11.1. Prognosis

Most patients with HS have mild compensated disease, and the sources consulted do not describe excess mortality attributable to uncomplicated HS; the risks determining long-term prognosis are severe infection after splenectomy, late vascular events, gallstones and iron overload [6, 11]. In the Israeli cohort, none of the 61 children with an aplastic crisis developed sustained marrow failure [9]. Splenectomy corrects anaemia but increases the risk of late venous (HR 3.3) and arterial (HR 7.2) events compared with affected individuals who have not undergone splenectomy, and OPSI risk persists throughout life [11, 74, 77]. In splenectomised US veterans (more than 8000, follow-up up to 27 years), sepsis and thrombosis risks persisted long term [77]. Partial splenectomy preserves splenic function in 96% of children, but half of severely affected children required completion splenectomy [11]. Severe recessive SPTA1 forms with almost complete α-spectrin deficiency have a prognosis dependent on transfusions, chelation and, exceptionally, transplantation [12, 42]. Life expectancy in xerocytosis is affected by iron overload and thrombosis, particularly after splenectomy; in the French series, the proportion treated for iron overload rose from 25% below age 20 to 73% above age 40 [18].

11.2. Surveillance

The surveillance recommendations come from consensus and case series, not from comparative surveillance studies. The intervals for biliary ultrasound differ between the BSH and GeneReviews, and meningococcal vaccine boosters differ between the CDC and the BSH; the choice should follow the national schedule and the severity of the patient's disease [2, 6, 77, 101]. Splenectomised patients should remain under surveillance for biliary symptoms when the gallbladder has been preserved, and patients with xerocytosis should be followed lifelong for iron overload, even without anaemia [6, 18]. The French PNDS (National Diagnosis and Care Protocol) intervals for biliary ultrasound and for the adult consultation add a third position to the divergence between the BSH and GeneReviews, and the protocol itself indicates annual ultrasound in the summary sheet and with Gilbert syndrome; in DHSt, the correlation between ferritin and tissue iron is poor below 1000 µg/L, which justifies MRI from 500 µg/L [8].

12. Summary and Final Considerations

Hereditary spherocytosis is the most common hereditary haemolytic anaemia in populations of Northern European ancestry, with an estimated prevalence of approximately 1:2000. It results from usually dominant ANK1, SPTB and SLC4A1 variants or recessive SPTA1 and EPB42 variants that weaken vertical links between the spectrin skeleton and the lipid bilayer. Membrane loss produces spherocytes that the spleen destroys, and clinical manifestations range from asymptomatic carriage to hydrops fetalis, including neonatal jaundice, anaemia with splenomegaly, parvovirus B19 aplastic crises and pigment gallstones. Diagnosis is clinical and laboratory-based in most patients; with a family history and a typical presentation, no other tests are needed, and in atypical cases combined EMA and AGLT detect almost all patients, with CDA type II and autoimmune haemolytic anaemia as the main pitfalls. Genetic testing has gained a role in severe, recessive and transfused forms and in uncertainty before splenectomy, but a negative panel does not exclude disease, particularly because of deep intronic variants and low-expression alleles such as αLEPRA.

Treatment is supportive: folic acid in moderate and severe forms, transfusions during crises and in the first year, and total or partial splenectomy in severe or transfusion-dependent forms, after 5–6 years whenever possible, laparoscopically, with vaccination, antibiotic prophylaxis, fever education and Doppler on day 7. Splenectomy corrects the anaemia, but carries a lifelong risk of severe infection and an increase in late vascular events, and is no longer routinely accompanied by cholecystectomy. The other membrane disorders change this logic: elliptocytosis is almost always asymptomatic, pyropoikilocytosis is its severe biallelic form, Southeast Asian ovalocytosis is a trait protective against malaria, and the stomatocytoses, in particular PIEZO1-related xerocytosis and KCNN4-related Gardos channelopathy, combine compensated haemolysis, iron overload, perinatal oedema and thrombosis, with splenectomy contraindicated. Targeted therapy is at an early stage, with mitapivat evaluated in a single-arm phase 2 trial and senicapoc without published results, and haematopoietic stem cell transplantation remains exceptional in refractory recessive forms. The 2021 French national protocol adds doses of neonatal erythropoietin and folic acid, thresholds for iron overload screening, French vaccination and antibiotic prophylaxis schedules and an age-based follow-up calendar, and diverges from the British and European guidelines on cholecystectomy for asymptomatic gallstones, partial splenectomy before 5 years and vaccine boosters.

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