Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)

INTRODUCTION — Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX) are rare disorders that cause variable hemolytic anemia and abnormal red blood cell (RBC) morphologies. Both are disorders of RBC hydration.

This topic discusses the mechanisms, evaluation, and management of stomatocytosis and xerocytosis.

Separate topics discuss general approaches evaluating hereditary hemolytic anemias:

●Anemia (child) – (See "Approach to the child with anemia".)

●Anemia (adult) – (See "Diagnostic approach to anemia in adults".)

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

●Spherocytosis – (See "Hereditary spherocytosis".)

●Schistocytes – (See "Non-immune (Coombs-negative) hemolytic anemias in adults", section on 'Fragmentation'.)

●Target cells, burr cells, and spur cells – (See "Burr cells, acanthocytes, and target cells: Disorders of red blood cell membrane".)

DEFINITIONS AND CLASSIFICATION — Stomatocyte and xerocyte are morphologic terms that describe the appearance of red blood cells (RBCs) on the peripheral blood smear.

●Stomatocytes (also called hydrocytes) contain a mouth-shaped area of central pallor on a stained blood smear (picture 1) and by scanning electron microscopy (picture 2).

●Xerocytes (also called dessicocytes) are markedly hyperchromic RBCs (picture 3).

These morphologic changes imply a pathophysiologic process. (See 'Pathophysiology' below.)

Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX) are autosomal-dominant inherited conditions with variable hemolysis and anemia [1]. In dehydrated HSt (also called stomatocytic xerocytosis) both abnormalities may be seen. Some syndromes with HSt cannot readily be distinguished from HX, and several intermediate forms exist. The traditional classifications are as follows:

●HSt – HSt has been classified into:

•Overhydrated HSt (OHS)

•Dehydrated HSt (DHS; synonymous with stomatocytic HX)

•Cryohydrocytosis (CHC)

•Familial pseudohyperkalemia (FP)

Additional forms of HSt with syndromic features include stomatin-deficient cryohydrocytosis with intellectual disability, seizures, and hepatosplenomegaly; phytosterolemia non-leaky stomatocytosis with macrothrombocytopenia; and dehydrated HSt with perinatal edema and/or pseudohyperkalemia [1]. (See 'Genetics' below.)

●HX – HX has been classified into [2]:

•Classic HX

•Stomatocytic HX (synonymous with dehydrated HSt)

•HX with hyperphosphatidylcholine hemolytic anemia

Genotype-phenotype correlations are increasingly reported, and the classification may benefit from incorporation of this information. (See 'Genetics' below.)

●SAO – Southeast Asian ovalocytosis (SAO) is a benign condition not associated with anemia and characterized by increased rigidity of the RBC membrane, with stomatocytes on the peripheral smear. SAO is discussed separately. (See "Southeast Asian ovalocytosis (SAO)".)

GENETICS — Classic HSt and HX are autosomal dominant [1,3,4]. De novo germline pathogenic variants are not uncommon; some children will have a negative family history [5].

The genotypes remain incompletely characterized. Some of the best understood gene variants affect red blood cell (RBC) membrane transporters such as PIEZO1, Gardos channel (KCNN4 gene), Rh-associated glycoprotein (RhAG, CD241), and SLC4A1 (band 3) [1]. In a series of 123 patients with HSt (49 independent pedigrees), nearly one-half of the families had pathogenic variants in PIEZO1 [6]. Some genotype-phenotype correlates have been observed, such as compensated hemolysis with PIEZO1 variants and more severe anemia and iron overload with KCNN4 variants [7]. (See 'PIEZO1' below and 'KCNN4 (Gardos channel gene)' below and 'RHAG' below and 'SCL4A1 (band 3 gene)' below and 'ABCB6' below.)

Additional rare syndromic forms of HSt include [1]:

●Pathogenic variants in the glucose transporter SLC2A1 can produce hemolytic anemia with stomatocytosis and cold-induced cation leak from the RBCs, as well as hepatosplenomegaly, cataracts, seizures, intellectual disability, and movement disorders [8,9].

●Pathogenic variants in ABCG5 and ABCG8, which encode two ATP-cassette transporters that mediate intestinal transport of dietary sterols, cause dyslipidemia, macrothrombocytopenia, and hemolysis with stomatocytosis [10-12]. The stomatocytosis is most likely due to altered incorporation of sterols in the RBC membrane.

PIEZO1 — Some cases of HSt and classic HX are caused by pathogenic variants in the PIEZO1 gene (also called FAM38A), which encodes pore-forming subunits of the PIEZO1 channel.

The word "piezo" comes from the Greek word "piesi," meaning pressure. PIEZO1 is a mechanosensitive channel capable of generating electrical current in response to mechanical tension [13,14]. The function of the channel in normal RBCs is unclear; it may play a role during RBC deformation in small vascular beds.

Several multigenerational kindreds of HX families with PIEZO1 variants have been reported [13,15-21]. These variants cause increased channel activity due to delayed channel inactivation and increased cation transport [13,18,19]. Identification of several novel PIEZO1 variants has produced a more complicated picture [19]; these new alleles may exhibit altered response to osmotic stress and impaired membrane trafficking, with some possibly resulting in increased RBC dehydration; they are not necessarily associated with delayed channel inactivation [19,22].

Many cases of HSt due to PIEZO1 variants are characterized by compensated hemolysis, perinatal edema (in approximately one-fifth of affected kindreds), and higher risk of thromboembolic complications post-splenectomy, especially portal thrombosis. In a study that evaluated clinical findings in 29 individuals with a pathogenic variant in PIEZO1, those with a variant affecting the pore domain of the ion channel had a more severe clinical phenotype; however, it was not possible to make extensive genotype-phenotype correlations [6].

Piezo1 is also expressed in liver and bone marrow during development, suggesting a correlation between pathogenic variants in PIEZO1 and the syndromic association of HX, perinatal edema, and pseudohyperkalemia [17]. PIEZO1-activating mutations lead to delayed erythroblast and reticulocyte maturation, possibly by mediating increased calcium entry into erythroblasts [23,24].

Certain variants in PIEZO1 may act as disease modifiers in other hematologic conditions [25,26]. As examples:

●Acute hemolysis and profound RBC dehydration have been described in an individual with hemoglobin C trait and a PIEZO1 variant [27].

●In a cohort of 20 beta thalassemia carriers, some individuals had anemia of varying degrees, splenomegaly, alterations in hemolysis indices, and liver hemosiderosis [28]. These individuals, referred to as symptomatic beta thalassemia carriers, were highly likely to have hereditary anemia variants; notably, approximately 93 percent of the symptomatic beta thalassemia carriers had pathogenic variants in the PIEZO1 gene.

It is also important to evaluate the possible co-inheritance of causative variants in genes related to hereditary stomatocytosis with those causing other RBC disorders. In a case series of 155 consecutive patients with various RBC disorders, 15 percent had multi-locus inheritance (more than one disease variant in each patient) [26]. This mainly involved variants in the PIEZO1 and SPTA1 genes causing hereditary spherocytosis.

Additionally, from the genetic perspective, PIEZO1 is a large and highly polymorphic gene that exhibits reduced genetic constraints. Consequently, in most cases, the American College of Medical Genetics and Genomics (ACMG) classification fails in assessing the pathogenicity of genetic variants within this gene. Therefore, accurate variant interpretation often necessitates specific knowledge about the patient, gene variant, or specific disease, making it crucial to conduct genetic testing for dehydrated HSt in specialized laboratories. (See "Genetics: Glossary of terms", section on 'Variant'.)

Certain variants in PIEZO1 appear to be protective against severe malaria, and a 2018 study determined that as much as one-third of the population in certain African countries carry a gain-of-function mutation (E756del) in PIEZO1 that does not cause clinical disease [29]. This allele is also present in approximately 14 percent of African Americans. In another study in individuals with sickle cell disease (SCD), this same allele has been associated with increased red blood cell density [30]. (See "Protection against malaria by variants in red blood cell (RBC) genes".)

The Er red blood group antigens are produced by amino acid substitutions in the extracellular domain of Piezo1 [31]. (See "Red blood cell antigens and antibodies", section on 'Er blood group system'.)

KCNN4 (Gardos channel gene) — KCNN4 encodes the Gardos channel, the "calcium-activated potassium channel of red blood cells" [32-35]. Each RBC contains approximately 120±36 Gardos channels [36]. No other type of RBC potassium channel has been demonstrated in human RBCs.

The Gardos channel is activated by micromolar concentrations of calcium (k₅₀ = 0.3 to 2 micromol/L) and inhibited by charybdotoxin, clotrimazole, other imidazole antimycotics, and senicapoc [37-39].

Some cases of dehydrated HSt (also called stomatocytic HX) are caused by pathogenic variants in KCNN4; these variants are generally associated with more severe anemia and hemolysis than HSt due to variants in PIEZO1 and more pronounced iron overload (estimated from serum ferritin values), with variable RBC dehydration.

Several kindreds with pathogenic variants in KCNN4 have been reported, including R352H (in the calmodulin-binding domain) and V282M/E (at the cytoplasmic face of the TM6 portion) [21,40-42]. The RBCs from affected patients have increased channel sensitivity to intracellular calcium that causes reduced cellular potassium and in turn leads to cellular dehydration and formation of xerocytes [40]. With more kindreds being described, more nuanced RBC phenotypes have emerged that suggest the presence of disease modifiers and/or secondary alterations of membrane function due to the profound dehydration and shrinkage of the RBCs [43,44].

Activation of the Gardos channel is also thought to contribute to RBC dehydration in sickle cell disease (SCD). Senicapoc, a high-affinity inhibitor of the Gardos channel, was evaluated in patients with SCD, but a randomized trial failed to show measurable clinical benefits compared with placebo [45]. (See "Investigational therapies for sickle cell disease", section on 'Increasing RBC hydration (senicapoc, memantine)'.)

The identification of several new variants that cause a constitutively active Gardos channel in individuals with HX and the role played by the Gardos channel in dehydration has led to renewed interest in senicapoc as a candidate for the treatment of HX [46,47].

RHAG — Overhydrated HSt is caused by pathogenic variants in RHAG, which encodes the Rh antigen-associated glycoprotein (RhAG, CD241) [48,49]. The associated Rh blood group antigen was originally identified using cells from Rhesus monkeys. Some citations retain this term, but the correct name is Rh. (See "Red blood cell antigens and antibodies", section on 'Rh blood group system'.)

RhAg is a NH₃/NH₄⁺ transporter; it has also been reported to control RBC gas exchange [4]. RhAG is a component of a macrocomplex with band 3. (See "Red blood cell membrane: Structure and dynamics", section on 'Band 3'.)

Heterozygosity for a missense mutation (F65S) in RHAG has been reported in the majority of patients with overhydrated HSt. This variant impairs the function of the channel and increases cation leak; the latter is thought to be an effect on other cellular permeability pathways [50].

SCL4A1 (band 3 gene) — SLC4A1 (for solute carrier) encodes band 3; the gene is also called BND3 or AE1 (for anion exchanger). Each RBC contains approximately 1.2 million copies of band 3 per cell.

HSt (typically, the cryohydrocytic form) can be caused by pathogenic variants in SLC4A1. One individual with HSt in association with dyserythropoiesis also had an SLC4A1 variant [51].

A specific genetic variant in SLC4A1 gene that deletes nine amino acids is responsible for Southeast Asian ovalocytosis (SAO); this condition is discussed separately. (See "Southeast Asian ovalocytosis (SAO)".)

Band 3 has two main functions:

●Anion exchange – The protein is an anion exchanger that passively transports bicarbonate ions out of the RBC in exchange for chloride (Cl^-) ions via the C-terminal membrane-spanning domain. Its physiological function is to facilitate removal or carbon dioxide generated from cellular respiration in the tissues in the form of bicarbonate that can be transported to the lungs [4]. This is related to its ability to bind deoxyhemoglobin, but not oxyhemoglobin, with high affinity [52]. This interaction also mediates a switch in glucose metabolism between the pentose phosphate and the glycolytic pathways, as well as ATP release from the RBC [53]. The bicarbonate ions are used to remove carbon dioxide produced from tissue respiration.

All of the SLC4A1 variants that cause HSt appear to interfere with anion transport [51,54-56]. HSt-associated variants are believed to turn the anion exchanger into an unregulated cation channel.

●RBC membrane-cytoskeleton structure – The protein links ankyrin to the RBC membrane via the N-terminal cytoplasmic domain, which binds the protein complex containing ankyrin, band 4.1, and band 4.2, as well as hemoglobin and glycolytic enzymes. (See "Red blood cell membrane: Structure and dynamics", section on 'Band 3'.)

SLC4A1 variants that interfere with the cytoskeletal functions can cause hereditary spherocytosis (HS) and Southeast Asian ovalocytosis (SAO) [57]. (See "Hereditary spherocytosis", section on 'Band 3 deficiency due to SLC4A1 variants' and "Southeast Asian ovalocytosis (SAO)".)

●Mitosis – Band 3 also regulates mitosis in erythropoietic cells and may be involved in dyserythropoietic disorders [58].

Absence of band 3 was thought to be incompatible with life due to severe hemolytic anemia [59]. However, band 3 variants have been described in which individuals homozygous for the variant have survived [60]:

●Band 3 Coimbra (Val488Met) – Homozygosity causes complete absence of band 3 in RBCs. A newborn with this variant had severe hemolysis, hydrops, distal renal tubular acidosis (RTA), and nephrocalcinosis and required chronic blood transfusion and daily bicarbonate [61,62]. Splenectomy resulted in transfusion independence for nine years, with regular transfusions plus iron chelation restarted at age 12. At age 19, the distal RTA was well controlled with normal kidney function [63].

●Band 3 Neapolis – This variant contains a T-to-C substitution at the +2 position in the donor splice site of intron 2, resulting in an 88 percent reduction of band 3 in RBCs and producing severe, transfusion-dependent hemolytic anemia that improved markedly after splenectomy [64].

●Band 3 null^VIENNA (S477X) – This variant contains a nonsense mutation that abolishes band 3 expression, associated with transfusion-dependent hemolysis, dyserythropoiesis, and complete distal RTA [63].

●Band 3 SAO variant – This variant deletes nine amino acid residues. It was originally thought to be incompatible with life, but a few individuals have been described with homozygosity for the variant. (See "Southeast Asian ovalocytosis (SAO)", section on 'Specific SLC4A1 deletion'.)

ABCB6 — HSt and pseudohyperkalemia has been seen with pathogenic variants in ABCB6, which encodes a porphyrin transporter that serves as the basis for the Langereis (Lan) blood group system [65,66]. (See "Causes and evaluation of hyperkalemia in adults", section on 'Pseudohyperkalemia'.)

A 2016 survey of 327 blood donations found evidence of an ABCB6 variant in one; after four weeks of storage, the RBCs showed massive loss of K⁺, and the authors suggested that neonates receiving large-volume transfusions of whole blood from a donor with this type of variant could develop hyperkalemia [67].

PATHOPHYSIOLOGY

Control of RBC solute and water content — The red blood cell (RBC) volume is controlled by changes in cations, anions, and cell water, which are continuously regulated by the activity and interactions of several ion transport systems, as summarized in the figure (figure 1) [68,69]. This includes two energy-drive systems (ATPases) and several passive (gradient-driven) transport systems.

The importance of these systems is indicated by the various diseases occurring when red cell ion transport is impaired (table 1).

●Cation transport

•Na-K-ATPase pump – The sodium-potassium (Na-K)-ATPase pump is an energy-driven pump that maintains an intracellular milieu low in Na and high in K. It extrudes three Na ions in exchange for two K⁺ ions. It has three subunits (alpha-1 catalytic subunit that transports ions, beta-1 subunit that binds membrane and cytoskeletal components, and gamma-1 proteolipid subunit) [70,71].

Each RBC contains approximately 228 to 470 Na-K-ATPase units [72].

The outward K⁺ gradient and the inward Na gradient is used by several passive transport systems that are sensitive to changes in pH, volume, or membrane integrity (the pump-leak model) [73].

•Ca-ATPase pump – The calcium (Ca)-ATPase pump is a powerful energy-driven pump that maintains low free cytosolic calcium levels (<0.1 µmol/L) [74]. The pump extrudes one Ca ion in exchange for one hydrogen ion at the expense of one ATP molecule.

Activity of the Ca-ATPase pump varies between RBCs in the same individual, with subpopulations of RBCs in which activity can vary six- to ninefold [75]. Normal RBC aging is associated with decreased Ca-ATPase activity [76].

•Na-Mg exchanger (SLC41A1 gene) – RBCs have a high total magnesium (Mg) but a less free Mg. Mg regulates several cellular functions; its export from RBCs is regulated by a gradient-driven Na-Mg exchanger (or antiporter, encoded by the SCL41A1 gene) that exports Mg in exchange for Na [77,78].

Mechanisms that control magnesium entry into RBCs are not well understood.

•Na-H exchanger (SLC9A1 gene) – The Na-hydrogen (H) exchanger (or antiporter, encoded by the SLC9A1 [also called NHE1] gene) transports Na into RBCs in exchange for H, accounting for a small fraction of the passive Na influx [79]. The exchanger is regulated by protein 4.1R; deletion in a mouse model causes spherocytosis [80,81]. (See "Hereditary spherocytosis", section on 'Pathophysiology'.)

In non-RBC cells, Na-H exchange regulates pH and mediates volume increase following hypertonic shrinkage.

•TRPC6 channels – Transient receptor potential channels of canonical type 6 (TRPC6) channels are responsible for the leak of Ca into RBCs [82]. They may become active in the terminal phase of the erythrocyte lifespan and help to initiate eryptosis (apoptosis-like death of erythrocytes) [83].

●Anion transport

•Band 3/AE1 (SLC4A1 gene) – Band 3 (anion exchanger 1 [AE1]) exchanges chloride and bicarbonate ions. (See 'SCL4A1 (band 3 gene)' above.)

•Na⁺-K⁺-Cl^- cotransporter (SLC12A gene) – The Na-K-Chloride (Cl) cotransporter is present on RBCs as well as numerous other cell types [84]. The major erythroid form extrudes one Na and one K⁺ with two Cl^- [85]. It plays a minor role in regulating RBC volume.

•K⁺-Cl^- cotransporter (SCL12A4 gene) – The K⁺-Cl^- cotransporter belongs to the family of chloride-cation transporters (CCC) that contains at least four major isoforms (KCC1-4) expressed in different cell types (RBCs express KCC1, 3, and 4) [86-89]. It extrudes K⁺ and Cl^-. It is activated by cell swelling and hydrostatic pressure, as well as by urea, oxidation, and positively charged hemoglobin variants [90-101]. It is inhibited by intracellular divalent cations including Mg and zinc [102].

●Water diffusion and transport – Water movement passively follows that of intracellular cations (Na⁺, K⁺) and anions (Cl^-) or is induced by changes in tonicity of the environment surrounding the RBC. Water can be transported more rapidly by aquaporin-1 (AQP-1, previously called CHIP28) [103,104].

The Colton blood group antigen polymorphism is a single amino acid substitution in an extracellular domain of AQP-1 [105]. Colton-null individuals lack AQP-1 and have a modest reduction in urinary concentrating ability [106,107]. (See "Red blood cell antigens and antibodies", section on 'Colton blood group system'.)

Normal RBC aging is accompanied by progressively increased cell density due to loss of K⁺ and water, until the terminal phase of the RBC lifespan, when dense RBCs gain Na, swell, and are removed from the circulation [108,109].

Stomatocyte formation — When the normal biconcave disc becomes a uniconcave cup (picture 4 and picture 2), a RBC will appear as a stomatocyte on the peripheral blood smear (picture 1).

In vitro studies have led to the proposal that relative expansion of the inner leaflet of the bilayer relative to the outer leaflet may be responsible [110]. This conclusion is based on studies in which RBC shapes can be modified by exposure to amphiphilic agents (compounds that can simultaneously interact with liquids [such as cytoplasm] and lipids [in membranes]). These compounds are thought to intercalate passively into the relatively negatively charged inner half of the membrane phospholipid bilayer, causing an inner bulging (the beginning of the cup) (figure 2) [110,111]. Endocytic vacuoles then form at the advancing mouth causing the loss of surface area that completes the transformation to a stomatocyte.

There are several mechanisms by which this change can occur:

●In HSt, the mechanism often involves changes in cell volume caused by reduced intracellular ion content. (See 'Control of RBC solute and water content' above.)

●In most acquired stomatocytosis and rare inherited conditions that affect lipid metabolism, the mechanism often involves a decrease in RBC membrane surface area or qualitative changes in the composition of the membrane lipid bilayer.

●In some healthy individuals, stomatocytes occasionally can be found due to a drying artifact; hence, it is important to evaluate several different areas of the peripheral smear before determining that a patient has circulating stomatocytes.

In contrast with expansion of the inner leaflet of the membrane, which is postulated to produce stomatocytes, expansion of the outer leaflet leads to echinocytes (burr cells). However, other theories postulate that alterations in structure and/or function of band 3 or other cytoskeletal protein may result in stomatocytic or echinocytic changes. (See "Burr cells, acanthocytes, and target cells: Disorders of red blood cell membrane", section on 'Causes of burr cells'.)

Alterations in ion content can also cause stomatocytosis. In disorders characterized by overhydration of the RBCs, stomatocytosis may represent a step in the transformation of a discocyte into an overhydrated spherocyte, which is followed by RBC fragmentation. In RBC dehydration disorders, stomatocytes may be a step in the generation of dehydrated spherocytes and/or echinocytes.

Xerocyte formation — Xerocytes are dense, hyperchromic RBCs. They form when loss of cell solutes induces osmotic water loss. (See 'Control of RBC solute and water content' above.)

In xerocytosis, potassium (K⁺), the main intracellular cation, is lost. This may be associated with a slight increase in intracellular sodium (Na⁺), but the net cation content of the RBC is significantly lower than in control RBCs.

When the leakage of K⁺ out of the cell exceeds the rate at which it is pumped back in, low intracellular K⁺ and dehydration ensue [5]. If the permeability of the membrane to Na⁺ is abnormally high and the Na⁺-K⁺-ATPase is unable to fully compensate, the cells gain Na⁺ with overhydration [5,112]. (See 'Control of RBC solute and water content' above.)

●Dehydration – Dehydration occurs with loss of intracellular cations (K⁺), which leads to osmotic water loss. Dehydrated stomatocytes have markedly decreased intracellular K⁺ with normal or increased intracellular Na⁺.

Dehydrated HSt (also called stomatocytic HX) has been linked to variants in the genes encoding the Piezo1 and Gardos channels. (See 'Genetics' above.)

●Overhydration – Overhydrated HSt is less common than dehydrated HSt. Overhydration occurs when cellular permeability to Na⁺ increases (cation leak into the cell), leading to a marked increase in intracellular Na⁺ that cannot be compensated for by the Na⁺-K⁺-ATPase pump [113]. The increased intracellular cation content causes an osmotic increase in cell water, leading to overhydrated HSt.

Overhydrated HSt may be caused by pathogenic variants in the RHAG gene, which controls cation flux. (See 'Genetics' above.)

●Cryohydrocytosis – Cryohydrocytosis is a rare form of overhydrated HSt associated with hemolysis of RBCs in vitro when the cells are refrigerated [114]. Cryohydrocytes are RBCs that have a mild cation leak at body temperature and a marked increase in cation permeability at refrigerator temperature [113]. Thus, they become overhydrated when refrigerated; they may also form stomatocytes or microspherocytes in vitro and/or leak sufficient K⁺ into the plasma in the collection tube to cause pseudohyperkalemia [5,114,115]. In some cases, pathogenic variants in SLC4A1, which encodes band 3, have been reported [116-119]. Two cases of stomatin deficiency associated with variants in SLC2A1 have been reported [9]. HSt associated with pseudohyperkalemia has been designated familial pseudohyperkalemia. (See 'Clinical manifestations' below and "Causes and evaluation of hyperkalemia in adults", section on 'Pseudohyperkalemia'.)

Dehydration also usually produces target cells, and pronounced dehydration produces xerocytes, which is reflected by a substantially increased mean corpuscular hemoglobin concentration (MCHC) (picture 3) [120]. Xerocytes have decreased osmotic fragility due to the small baseline cell size. Xerocytes also exhibit a characteristic increase in cell rigidity and reduction in deformability, changes that are thought to promote hemolysis. (See 'Effects on the RBC (hemolysis, metabolic abnormalities, membrane changes)' below.)

Effects on the RBC (hemolysis, metabolic abnormalities, membrane changes) — The precise mechanisms leading to hemolysis are unknown. A reduction in deformability leading to increased sensitivity to mechanical or shear stress is believed to be a unifying mechanism of hemolysis for all types of stomatocytosis. Hemolysis is mostly extravascular, although there may be an intravascular component in severe cases. Intravascular hemolysis was reported in a patient with HX during intensive exercise [121].

In some patients, anemia is absent and hemolysis can be observed only in particular conditions associated with increased mechanical stress to the RBCs [15,121]. (See 'Compensated hemolysis and hemolytic anemia' below.)

Reduced deformability has been quantified in the research setting using techniques such as osmotic gradient ektacytometry, but the results do not necessarily predict the behavior of the RBCs in vivo. Less deformable RBCs are believed to be predisposed to trapping in the microvasculature of the spleen and other organs of the reticuloendothelial system, which can lead to increased phagocytosis and varying degrees of extravascular hemolysis.

In addition to hemolysis, overhydrated HSt RBCs have a distinct set of metabolic abnormalities, alterations in adherence to the endothelium, and a deficiency in the stomatin protein.

●Metabolic – Metabolic abnormalities in RBCs of patients with overhydrated HSt include increased adenosine diphosphate (ADP), reduced 2,3-bisphosphoglycerate (2,3-BPG, previously called 2,3-DPG), and enhanced oxygen affinity [122,123]. Many of these findings are consistent with increased ATP production, which is needed to support the increased activity of the Na⁺-K⁺-ATPase required to compensate for the increased Na⁺ permeability. Other changes include increases in oxidized glutathione efflux, accumulation of glutamine and tryptophan, abnormal phosphatidylserine externalization, and decreases in creatine transport and concentration of ergothioneine, but the mechanism is not clear [40].

●Membrane – RBC membrane abnormalities have been reported in various forms of HSt and HX. It is unclear whether these have any causal roles in hemolysis or other clinical manifestations.

•RBCs from some patients with HSt have increased adherence to endothelium [124]. The abnormal stickiness may mediate thromboembolic complications seen in these patients, particularly after splenectomy. (See 'Other findings' below.)

•RBCs from some patients with overhydrated HSt were deficient in the membrane protein stomatin (also known as band 7.2b) [3,125-127]. The function of stomatin is uncertain, but studies in mice suggest its deficiency alone does not cause stomatocytosis or hemolysis [13,15,16].

•RBCs from some patients with HX have a relative increase of membrane phosphatidylcholine, leading to the designation of "HX with hyperphosphatidylcholine hemolytic anemia" [2,116,128-130]. This abnormality is accompanied by an increase in membrane permeability to K⁺ and cellular dehydration.

EPIDEMIOLOGY — HSt and HX are rare. The incidences were estimated from a 2015 United Kingdom series [5]:

●Dehydrated HSt (stomatocytic HX) – 100 per 1 million births (1 in 10,000)

●Overhydrated HSt – 1 per 1 million births

●Cryohydrocytosis (a form of HSt) – Extremely rare

●Familial pseudohyperkalemia (a form of HSt) – Extremely rare

By comparison, the incidence of hereditary spherocytosis is >200 to 300 per 1 million births. (See "Hereditary spherocytosis", section on 'Disease prevalence'.)

A greater incidence of mild HX has been proposed based on a laboratory analysis of routine complete blood counts (CBCs) that showed a higher-than-expected proportion of individuals with possible HX based on the finding of an increased mean corpuscular hemoglobin concentration (MCHC) [131]. (See 'Diagnosis' below.)

CLINICAL MANIFESTATIONS

Typical presentations — HSt and HX can be asymptomatic or can produce chronic hemolytic anemia of varying severity. The most common presentation is of compensated hemolysis or mild anemia, high reticulocyte count, and elevated mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular hemoglobin (MCH). The mean corpuscular volume (MCV) can show mild macrocytosis due to increased reticulocytes. Chronic fatigue is often present in the absence of significant anemia.

The age of presentation depends on the specific variant as well as other inherited conditions and environmental factors.

●In neonates, the physical examination is generally unremarkable, although it may show pallor, jaundice, and/or ascites in a severely affected neonate. Prenatal and/or perinatal edema has been reported, which in rare cases has been associated with life-threatening hydrops fetalis. Edema usually disappears spontaneously before birth or a few months thereafter [132-134].

●In many cases, diagnosis is made in early adulthood, since many individuals do not have symptoms related to anemia, and symptoms related to other complications (gallstones, iron overload, splenomegaly) occur later. Often the appearance can be attributed solely to iron overload, as exemplified by carriers of the PIEZO1 E756del variant (associated with malaria resistance), or in other instances where dehydrated HSt was masquerading as primary hemochromatosis [6,29,135,136].

●The increasing use of the complete blood count (CBC) in asymptomatic individuals has resulted in earlier diagnosis in some individuals who otherwise might never have come to medical attention. In many individuals, an elevated reticulocyte count, MCHC, or MCH with normal hemoglobin prompts additional investigations leading to the diagnosis of HSt or HX. Evidence of hemolytic anemia may be present, but there is rarely a history of need for transfusions. Some patients may be misdiagnosed as having hereditary spherocytosis (HS) and are only correctly diagnosed as having HSt after the occurrence of thrombotic complications post-splenectomy. (See 'Splenectomy' below.)

Two large case series summarize the likelihood of different clinical features:

●In a series of 123 individuals with HSt (49 families), the predominant features were mild macrocytic anemia (mean hemoglobin, 12.5; mean MCV, 99.1), splenomegaly, gallstones, and increased ferritin [6]. The mean age at diagnosis was 21 years for individuals with PIEZO1 variants and 47 years for ABCB6 variants.

●In a series of 126 individuals with HX (64 families), the predominant features were hemolysis that persisted following splenectomy, gallstones, splenomegaly, and high serum ferritin (mean ferritin, 764 ng/mL) [137]. Anemia was absent in most (median hemoglobin, 13.1) and none required regular transfusions. The median age at diagnosis of the probands was 32 years (range, birth to 88 years); 11 were diagnosed before the age of 1 year. PIEZO1 variants were associated with compensated hemolysis, perinatal edema, and post-splenectomy thrombosis, especially in the portal system.

Compensated hemolysis and hemolytic anemia — HSt and HX are characterized by compensated hemolysis or hemolytic anemia and abnormal red blood cell (RBC) morphologies on the peripheral blood smear. For some, chronic hemolytic anemia may be exacerbated by concomitant viral infections and sometimes transient aplastic crises.

An exception is familial pseudohyperkalemia, which is an asymptomatic carrier state that is not associated with clinical hemolysis [5].

As noted above, fatigue may be out of proportion to the degree of anemia (see 'Typical presentations' above). The severity of fatigue is attributed to the left-shift in the oxyhemoglobin dissociation curve. (See "Structure and function of normal hemoglobins", section on 'Oxygen affinity'.)

●Hemolysis – Typical findings include elevated reticulocyte count, increased lactate dehydrogenase (LDH) and bilirubin, and low haptoglobin. The direct antiglobulin test (DAT; Coombs test) is negative.

Some individuals have chronic hemolysis, and others may have no hemolysis at baseline but may develop hemolysis in settings of stress such as infections or exercise. An example of the latter was reported in a competitive freestyle swimmer with HX, who had normal hemoglobin levels at rest but developed intravascular hemolysis and xerocytosis following exercise; he and his two affected sons were subsequently shown to carry the R2488Q PIEZO1 mutation [15,121]. Since xerocytes are more sensitive to shear stress, the increase in hemolysis with exercise may have resulted from a shift of blood flow to vessels in exercising muscle, which have smaller diameters and higher shear rates.

●Anemia – Individuals with HSt or HX have varying degrees of hemolytic anemia. The severity of anemia depends on the magnitude of hemolysis and the robustness of compensatory reticulocytosis. Some individuals may have severe hemolytic anemia, others may have evidence of compensated hemolysis, with reticulocytosis sufficient to maintain a normal hemoglobin/hematocrit despite ongoing hemolysis, and others may have no evidence of hemolysis [138].

In neonates presenting with hemolytic anemia, the severity may be great enough to necessitate neonatal exchange transfusion. Perinatal cases with severe intrauterine ascites or hydrops fetalis have also been described [118,119,132,133,139]. While most of the cases of nonimmune hydrops fetalis do not have a clear genetic basis, an activating PIEZO1 mutation is considered diagnostic of a genetic variant [140].

In a large case series that included 73 individuals with HSt, the mean hemoglobin was 12.5 g/dL and the mean corpuscular volume (MCV) was 99 fL [6]. In another case series, two-thirds of the patients had compensated hemolysis [7].

In some cases, the severity of anemia may suggest a specific genetic disorder. As examples, milder anemia and compensated hemolysis may indicate a PIEZO1 variant, whereas more severe anemia may indicate a KCNN4 variant. If a familial gene variant is not known, this information can be used to ensure that genetic testing includes the gene(s) most likely to be affected. (See 'Role of testing/which tests to perform' below.)

●RBC morphology – The blood smear shows stomatocytes (picture 1) in HSt and xerocytes (picture 3) in HX. In some cases, both morphologies may be present, and in others, there may be additional abnormalities such as target cells.

The percentage of stomatocytes may be as high as 40 to 60 percent in individuals with HSt [112]. The degree of stomatocytosis does not appear to correlate with the severity of hemolytic anemia [112].

Iron overload — Iron overload is frequently present. Therapies to address iron overload may be appropriate, especially in the presence of other conditions promoting iron overload such as hereditary hemochromatosis and/or a large number of transfusions [6,15]. (See 'Management' below and "Iron chelators: Choice of agent, dosing, and adverse effects".)

Other findings — HSt or HX can lead to consequences of hemolysis such as splenomegaly, pigmenturia, and/or pigmented gallstones [20].

Additional complications such as vaso-occlusion and/or thrombosis appear to be related to interactions between stomatocytes and the vasculature. These are more common following splenectomy, presumably because more stomatocytes remain in the circulation [65,117]. (See 'Management' below.)

●Vaso-occlusion – Some patients with HSt develop acute vaso-occlusive episodes similar to those seen in sickle cell disease (SCD) (see "Overview of the clinical manifestations of sickle cell disease", section on 'Vaso-occlusive pain'). The symptoms may include dyspnea, chest pain, or abdominal pain and are thought to result from enhanced adherence of stomatocytes to vascular endothelium [124]. This problem appears to be more common after splenectomy, perhaps because more stomatocytes remain in the circulation.

●Thrombosis – Some patients with HSt develop thrombosis, which may lead to pulmonary hypertension or other chronic complications. Thrombotic complications are frequently reported following splenectomy [141]. One series of nine patients with HSt described severe thrombotic complications following splenectomy in four (pulmonary hypertension in three and portal hypertension in one) [65].

Additional findings related to the genetic defect may occur:

●Pseudohyperkalemia – Some individuals with dehydrated HSt/stomatocytic HX have pseudohyperkalemia (an artifactual increase in serum K⁺ following storage of the blood sample at low temperature). This is a heterogenous disorder associated with varying degrees of hemolytic anemia or no anemia at all [8,142,143]. The finding of pseudohyperkalemia may be the only abnormality in some individuals with mild cryohydrocytosis. (See "Causes and evaluation of hyperkalemia in adults", section on 'Pseudohyperkalemia'.)

The finding of pseudohyperkalemia (rather than true hyperkalemia) can be verified by measuring K⁺ levels in paired samples, one kept at body temperature and the other at refrigerator temperature or on ice; in pseudohyperkalemia, the warm sample will show a normal K⁺ level and the cold sample will show hyperkalemia. Once the artifactual nature of the hyperkalemia is established, no further intervention for this finding is needed, although it is useful to note in the medical record that blood should not be refrigerated prior to testing. A search for the underlying genetic cause is of academic interest but not clinically relevant unless it is required for genetic testing.

●Neurologic problems – Reports have described cases in which HSt was associated with congenital intellectual disability, seizures, and cataracts; the gene variants responsible are not known [144]. (See 'Genetics' above.)

●Thrombocytopenia – A syndromic form of HSt is associated with macrothrombocytopenia and abnormal bleeding [1]. (See 'Genetics' above.)

EVALUATION

Diagnostic workflow — Typically, the diagnostic workflow for these conditions involves three lines of investigations:

●Initial evaluations that would lead to consideration of the diagnosis, including the complete blood count (CBC), blood smear review, and review of the family history and transmission pattern. (See 'When to suspect the diagnosis' below.)

●Biochemical testing and specialized analyses to identify the disorder.

●Molecular testing, typically done using a next-generation sequencing (NGS) approach, including targeted NGS gene panels or whole exome sequencing [145].

When to suspect the diagnosis — HSt or HX may be suspected in individuals with any of the following:

●Positive family history of HSt or HX, especially in a first-degree relative

●History of unexplained neonatal anemia and hyperbilirubinemia

●Non-immune hemolysis for which the cause remains unknown after routine initial testing

●Otherwise unexplained stomatocytes or xerocytes on the blood smear

●Elevated mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular hemoglobin (MCH) without another explanation

●Elevated ferritin and transferrin saturation (TSAT) without another explanation

Findings consistent with non-immune hemolysis include an increased reticulocyte count (unless there is a concomitant cause of anemia that impairs red cell production), increased lactate dehydrogenase (LDH) and bilirubin, and decreased haptoglobin. The direct antiglobulin test (DAT; Coombs test) is negative; one exception is following a recent transfusion that led to generation of an alloantibody. Anemia may be present, but if compensation is adequate, the hemoglobin value may be normal or only mildly decreased. (See "Overview of hemolytic anemias in children" and "Diagnosis of hemolytic anemia in adults".)

For individuals who do not have hemolytic anemia but for whom the diagnosis is suspected for one of the other reasons listed above, there may be benefits to making the diagnosis that include avoiding other extensive evaluations, avoiding splenectomy for other reasons, monitoring for signs of iron overload, and/or providing genetic counseling. The information may also be helpful if the patient is hospitalized for an acute illness and develops hemolytic anemia due to the stress of the illness.

Initial testing — Evaluation for HSt and HX includes the following:

●CBC – Review the complete blood count (CBC) and peripheral blood smear, which may show stomatocytes (picture 1) and/or xerocytes (picture 3), as well as target cells, schistocytes, or eccentrocytes. The stomatocyte shape in three dimensions is illustrated in the electron microscopy images (used for research) (picture 2).

•The blood smear should be reviewed closely to ensure there are no abnormalities of white blood cells (WBCs) or platelets; WBC and platelet counts are normal in HSt and HX.

•The red blood cell (RBC) indices typically show an increased mean corpuscular volume (MCV) of up to 140 femtoliters (fL) and abnormal MCHC (decreased in overhydrated HSt [typically 24 to 28 g/dL], increased in dehydrated HSt [typically 35 to 37 g/dL]) (figure 3) [5]. The impedance method used by Coulter instruments to measure MCHC underestimates the MCHC of dehydrated cells; as a result, diagnosis of HX may be missed unless instruments that directly measure MCHC are used [115]. MCH is frequently elevated as well, possibly due to the concomitant iron overload. (See "Approach to the child with anemia", section on 'Red blood cell indices' and "Diagnostic approach to anemia in adults", section on 'RBC indices'.)

The algorithm summarizes an approach to the evaluation based on findings from the CBC and blood smear (algorithm 1).

●Rule out other anemias – It is appropriate to confirm the initial findings and the lack of an immune mechanism and in some cases to rule out hemoglobinopathy or a microangiopathic process before proceeding to more specialized testing. This typically involves documenting a negative DAT and confirming that other concerning findings such as schistocytes and thrombocytopenia are absent.

In some cases, hemoglobin analysis (eg, by high-performance liquid chromatography [HPLC]) may be reasonable. Measurement of plasma potassium (K⁺) may be helpful if it shows pseudohyperkalemia, but absence of this finding is not informative [5]. (See 'Other findings' above.)

Southeast Asian ovalocytosis (SAO) has stomatocytic cells on the blood smear, but other differences help distinguish SAO from HSt and HX. (See 'Other causes of stomatocytosis' below and "Southeast Asian ovalocytosis (SAO)", section on 'Evaluation'.)

●Family history – Family history and transmission patterns should also be reviewed. Classic HSt and HX are autosomal dominant. Some individuals may have a de novo variant and a negative family history. (See 'Genetics' above.)

Our approach is consistent with a 2015 Guideline that addresses diagnostic testing for individuals with non-immune hereditary RBC membrane disorders [5].

In individuals for whom this testing is suggestive of HSt or HX, additional testing may include osmotic fragility and/or genetic analysis for possible gene variants associated with HSt and/or HX. (See 'Osmotic fragility or ektacytometry' below and 'Genetic testing' below.)

Osmotic fragility or ektacytometry

●Osmotic fragility – Osmotic fragility testing is generally performed after consultation with the hematologist or other expert in evaluation of anemias.

Interpretation of the results is as follows:

•Decreased osmotic fragility (non-incubated) and decreased RBC K⁺ and total cation content is seen in dehydrated HSt.

•Increased osmotic fragility (non-incubated) and increased RBC Na⁺ and total cation content is seen in overhydrated HSt [146].

•Autohemolysis at 4°C (greater at pH 8 than at pH 7.6 to 7.4) is seen in cryohydrocytic HSt [146,147].

•Stability to hemolysis upon exposure to high temperature (46 and 49°C for 15 to 60 minutes) and decreased K⁺ and total cation content is seen in HX [114].

Differences between the results in HSt, HX, and hereditary spherocytosis (HS) are discussed below. (See 'Differential diagnosis' below.)

●Ektacytometry – Osmotic gradient ektacytometry is an alternative technique used in Europe that provides a reliable diagnosis of the disease due to its unique pattern in HSt and HX [1,42,137,148].

•HX (dehydrated HSt) – In HX due to variants in PIEZO1, there is a leftward shift of the minimum in the deformability index (Omin) at low osmolarities, accompanied by a decrease in DI max [1]. For HX due to variants in KCNN4, the curve is normal.

•Overhydrated HSt – There is a rightward shift in the osmolarity curve [1].

•SAO – There is a unique pattern in which RBCs are completely non-deformable, resulting in a nearly flat curve across a wide osmotic gradient.

•Cryohydrocytosis and familial pseudohyperkalemia – The findings in these two conditions are less well studied. One report of cryohydrocytosis reported a pattern similar to hereditary spherocytosis in sodium-containing buffer, with a leftward shift, whereas in cold potassium-containing buffer, there was an extremely abnormal pattern with the entire curve shifted rightward [149].

Ektacytometry is also available in the United States in a limited number of laboratories. (See 'Resources for testing' below.)

●RBC cation content measurements – These may be obtained for research but are not available as a clinical test.

DIAGNOSIS — The diagnosis of HSt or HX is typically made by demonstrating anemia and/or chronic hemolysis associated with the characteristic changes in RBC morphology (stomatocytosis and/or xerocytosis) in conjunction with altered RBC indices such as elevated mean corpuscular hemoglobin concentration (MCHC), elevated mean corpuscular volume (MCV), and diagnostic changes on osmotic fragility testing or ektacytometry (algorithm 1).

The diagnostic workflow is described above. (See 'Evaluation' above.)

Up to one-third of patients do not have overt hemolysis, and lack of hemolysis does not eliminate the possibility of HSt or HX, especially if other features suggest one of these diagnoses [1,112,146,150].

In some cases, genetic testing may be used to establish the diagnosis, especially if a familial variant is known. If diagnostic changes are found on osmotic fragility testing or ektacytometry, genetic testing can be confirmatory but is not required. (See 'Genetic testing' below.)

GENETIC TESTING

Role of testing/which tests to perform — The use of genetic testing is individualized based on the findings from red blood cell (RBC) osmotic fragility testing (to confirm the diagnosis if needed), as well as the clinical severity, family history, and possible need for prenatal testing of family members [1,47].

However, genetic testing is not required for diagnosis, especially in patients who are asymptomatic and/or do not have evidence of ongoing hemolysis.

Some affected children will have a negative family history, and many of these will be found to have a new germline variant [5]. Thus, family history is more helpful if positive but cannot be used to exclude the diagnosis if negative. (See 'Genetics' above.)

The appropriate test depends on the clinical scenario (familial variant known, which disorder is suspected):

●Known familial variant – When the familial variant is known, genetic testing can confirm the presence or absence of the variant in other family members. This is especially true for well-characterized variants with established pathogenicity. In these cases, the familial variant can be tested without the need for more extensive testing.

●Suspected dehydrated HSt and HX – For suspected dehydrated HSt and HX, PIEZO1 variants should be assessed, although some patients with these conditions may have negative DNA analysis. In PIEZO1 variant-negative cases, Gardos channel (KCNN4) variants should be investigated.

●Suspected overhydrated HSt – For suspected overhydrated HSt, testing should include RHAG/SLC4A1 and SLC2A1.

In some cases, a gene panel test may be more economical than single gene testing; if this is used, it is important to confirm that the desired gene(s) and variants in those genes are on the panel. (See "Genetic testing", section on 'Extent of DNA analysis'.)

If a new variant is identified and its pathogenicity is unknown, it generally must be evaluated functionally in a specialized research laboratory that has access to techniques such as patch clamp testing and cell culture. This is especially relevant for new PIEZO1 variants, which exhibit a high degree of genetic polymorphism [7,151,152].

Genetic testing may also be appropriate for some individuals who are considering splenectomy, especially if there is any doubt about the diagnosis. However, this testing may not be available for some patients.

Genetic testing of family members — We prefer to test all first-degree relatives of an individual with HSt or HX, if possible. Potential benefits of genetic testing include the ability to distinguish between affected and unaffected individuals in a family and identification of those who may require closer monitoring for hemolysis, especially at times of increased stress (eg, infections, hospitalizations), those who must avoid splenectomy, and those who may be at risk of iron overload due to the presence of the HFE C282Y variant.

In addition, information about the genetic defect may be useful for future enrollment in clinical trials and/or for use of investigational or newly developed therapies that target a specific genetic defect.

General discussions of genetic counseling for family members and genetic testing of children are available separately. (See "Genetic counseling: Family history interpretation and risk assessment" and "Genetic testing", section on 'Ethical, legal, and psychosocial issues'.)

Resources for testing — The following laboratories/experts serve as reference laboratories for the diagnosis of rare anemias including HSt and HX:

●United States

•Blood Disease Reference Laboratory, Department of Pathology

310 Cedar Street, LH215

New Haven, CT 06520

Tel: 203-785-4492

Fax: 203-785-3896

https://medicine.yale.edu/pathology/clinical/mdx/

Medical Director: Pei Hui, MD, PhD

Laboratory Supervisor: Teresa Silva, e-mail: [email protected]

•Cancer and Blood Diseases Institute Erythrocyte Diagnostic Laboratory

Cincinnati Children's Medical Center

3333 Burnet Avenue, R1553

Cincinnati, OH 45229-3039

Tel: 513-636-4234

Fax: 513-636-2106

https://www.cincinnatichildrens.org/service/c/cancer-blood/hcp/clinical-laboratories/erythrocyte-diagnostic-lab

E-mail: [email protected]

Medical Directors: Charles T Quinn, MDS and Theodosia Kalfa, MD, PhD

Laboratory Supervisor: Mary Reynaud, e-mail: [email protected]

●Europe – An updated list of investigators and laboratories can be found on websites such as:

•European Network for Rare and Congenital Anemias (ENERCA)

enerca.org

•Orphanet portal

https://www.orpha.net/consor4.01/www/cgi-bin/Clinics.php?lng=IT

Specific laboratories in Europe that perform ektacytometry include:

•CEINGE biotecnologie avanzate Franco Salvatore, Naples Italy (Proff. A. Iolascon, I. Andolfo, R. Russo)

Email: [email protected]

https://www.ceinge.unina.it/index.php/bioingegneria

•Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico, Milan, Italy (Dr. P. Bianchi)

Email: [email protected]

https://www.policlinico.mi.it/i-nostri-professionisti/profilo/1015/bianchi-paola

•University Medical Center Utrecht, Netherlands (Prof. R. Van Wijk)

Email: [email protected]

https://www.umcutrecht.nl/en/research/researchers/van-wijk-richard-ha#

•INSERM U1134, INTS, Paris, France (Dr. Loïc Garçon)

DIFFERENTIAL DIAGNOSIS — The differential diagnosis of HSt and HX includes a variety of other inherited and acquired conditions that cause hemolytic anemia and/or red blood cell (RBC) morphologic abnormalities.

Hemolytic anemias — There are numerous other causes of hemolytic anemia. The most likely causes differ by age, ethnic background, and presence of other medical conditions. The figure shows a conceptual framework for these disorders (figure 4).

●Hereditary enzyme disorders – Some hemolytic anemias are inherited RBC enzymatic deficiencies such as glucose-6-phosphate dehydrogenase (G6PD) deficiency or pyruvate kinase (PK) deficiency. Less common RBC enzyme disorders are also possible. (See "Diagnosis and management of glucose-6-phosphate dehydrogenase (G6PD) deficiency" and "Pyruvate kinase deficiency" and "Rare RBC enzyme disorders".)

●Hereditary spherocytosis – Membrane/cytoskeletal disorders include hereditary spherocytosis (HS) and others. In addition to morphologic differences, there are differences in the genetics and specialized testing. As an example, HS is associated with gene variants affecting band 3 and others. RBCs in HS show increased (incubated) osmotic fragility, as well as decreased eosin-5-maleimide (EMA) binding and a positive acidified glycerol lysis (AGLT) test for lysis in <900 seconds. In contrast, in most cases of HSt, EMA binding is normal or increased and the AGLT is negative [5]. (See "Hereditary spherocytosis".)

●CDAs – Congenital dyserythropoietic anemias (mainly CDAI and CDAII) are discussed separately. (See "Overview of causes of anemia in children due to decreased red blood cell production", section on 'Congenital dyserythropoietic anemia'.)

●Acquired disorders – Other disorders are acquired; some may be immune mediated, such as hemolytic disease of the fetus and newborn (HDFN) or paroxysmal nocturnal hemoglobinuria (PNH). (See "Alloimmune hemolytic disease of the newborn: Postnatal diagnosis and management" and "Clinical manifestations and diagnosis of paroxysmal nocturnal hemoglobinuria".)

Like HSt and HX, these conditions may have anemia and laboratory findings consistent with hemolysis. Unlike HSt and HX, they typically have differing RBC morphologies on the peripheral blood smear (in HS, predominance of spherocytes rather than stomatocytes or xerocytes), and specialized testing in these other conditions will reveal the alternative diagnosis.

Other causes of stomatocytosis — A number of other inherited and acquired disorders may be associated with stomatocytes on the peripheral blood smear. These conditions are distinguished from HSt by review of the clinical scenario and specialized laboratory testing. An approach to determining the cause of stomatocytosis or a high MCHC is illustrated in the algorithm (algorithm 1).

●Hereditary

•SAO – Southeast Asian ovalocytosis (SAO) is a heritable RBC disorder caused by pathogenic variants affecting band 3; it is most frequently seen in individuals from parts of Southeast Asia, including Malaysia, New Guinea, Indonesia, and the Philippines. Like HSt, in SAO the cells have an oval or elliptocytic shape on the blood smear, often with one or two longitudinal slits. Unlike HSt, in SAO there is a specific deletion in the SLC4A1 gene, and hemolytic anemia typically is not seen after three years of age in individuals with SAO. (See "Southeast Asian ovalocytosis (SAO)", section on 'Clinical features'.)

•Rh null disease – Complete deficiency of Rh antigens on the RBC surface (Rh null disease) causes hemolytic anemia of variable clinical severity, stomatocytosis, and, with continuing loss of membrane, hyperdense spherocytes with increased osmotic fragility. Patients may be identified during pretransfusion testing. The diagnosis is made by demonstrating that all antigens of the Rh system are absent from the RBC surface [153]. (See "Red blood cell antigens and antibodies", section on 'Rh blood group system'.)

•Tangier disease – Tangier disease is an autosomal recessive disorder associated with a derangement in cholesterol metabolism, characterized by hypertriglyceridemia, very low levels of high-density lipoprotein, and increased deposition of cholesterol esters in tissues and in phagocytic cells [154]. Tangier disease may be associated with hemolysis, and in one patient, was characterized by stomatocytosis and increased osmotic fragility [155]. The abnormal membrane lipids may be responsible for the stomatocytosis, as described below for stomatocytosis in the setting of acquired stomatocytosis. (See "HDL cholesterol: Clinical aspects of abnormal values", section on 'Inherited causes'.)

●Phytosterolemia – Phytosterolemia (also called sitosterolemia or Mediterranean stomatocytosis/macrothrombocytopenia) is a rare inherited metabolic condition that affects intestinal absorption of cholesterol and plant sterols [10,156,157]. This in turn alters the membrane composition of RBCs as well as other cells such as platelets. Patients may have hemolytic anemia, stomatocytosis, and thrombocytopenia with increased platelet volume [5,158]. (See "Lipoprotein classification, metabolism, and role in atherosclerosis", section on 'Exogenous pathway of lipid metabolism'.)

●Acquired

•Liver disease/alcohol – Stomatocytes can be seen with chronic liver disease (most often due to excess alcohol use) or acute alcohol intoxication [159]. The stomatocytosis with acute alcohol intoxication appears to be transient, and it may affect a significant proportion of RBCs [159,160]. In one series of 100 patients admitted to a general medical ward with alcohol intoxication, 15 percent had ≥10 percent stomatocytes and another 29 percent had 5 to 9 percent stomatocytes on the peripheral blood smear [159]. The mechanism is thought to be due to a reduction in RBC membrane surface area rather than an increase in RBC volume. (See 'Stomatocyte formation' above.)

•Medications – Some medications can cause transient stomatocytosis [161]. One study demonstrated formation of stomatocytes upon exposure of RBCs to the chemotherapeutic drug vinblastine and the antipsychotic agent chlorpromazine [162]. Intercalation of the drug into the inner half of the lipid bilayer may be responsible for creating the abnormal morphology. (See 'Stomatocyte formation' above.)

•In vitro artifact – Stomatocytosis also can result as a drying artifact or peripheral blood smear preparation. (See 'Stomatocyte formation' above.)

Other causes of xerocytosis — Several other conditions are associated with xerocytes (or dense, hyperchromic cells resembling xerocytes) on the peripheral blood smear, with an increased mean corpuscular hemoglobin concentration (MCHC). These include hereditary elliptocytosis (HE) and hemoglobinopathies such as sickle cell disease (SCD) and homozygous hemoglobin C disease (Hb CC). In contrast, thalassemia is not associated with xerocytes; in thalassemia, the MCHC is reduced rather than increased.

●SCD – Cell dehydration is a characteristic feature of the RBCs in sickle cell disease (SCD) [163,164]. Morphology of the cells differs from xerocytes because of the prominent sickled shape (picture 5), but the changes in cellular properties show some overlap with xerocytes. The mechanism of cell dehydration in SCD involves loss of intracellular K⁺ due to alterations in K⁺-Cl^- transport and/or alterations in the Ca⁺⁺-sensitive K⁺ channel (Gardos channel) [165,166]. This is most likely due to interaction of sickle hemoglobin (Hb S) with the cell membrane or the regulatory machinery of the transporter [99]. These dense, dehydrated cells play an important role in pathogenesis of the vaso-occlusive manifestations of the disease [167-170]. (See "Pathophysiology of sickle cell disease", section on 'Membrane damage'.)

●Hb CC – Cell dehydration is a characteristic feature of homozygous Hb C (Hb CC) disease, which in most cases is characterized by a mild, compensated hemolysis with no significant anemia. The blood smear may show dehydrated cells and target cells (picture 6). Dehydration may be related to abnormal cell K⁺ loss via K⁺-Cl^- cotransport as well as a possible interaction between the abnormally positively charged hemoglobin protein and the cell membrane [90,99,101,171,172]. (See "Hemoglobin variants including Hb C, Hb D, and Hb E", section on 'Hb C'.)

Infantile pyknocytosis — Pyknocytes are hyperdense, dehydrated RBCs with irregular borders and varying numbers of projections. Infantile pyknocytosis is a condition that occurs in neonates, characterized by transient, non-immune hemolytic anemia that resolves in infancy. The cause (and whether it is due to a genetic or environmental factor) is unknown [173,174].

In one series that analyzed blood samples from 149 neonates with unexplained hemolytic anemia, pyknocytes were seen in 14 (9 percent) [175]. Pyknocytes may also be seen in individuals with HE, in whom the elliptocytes may not appear until later in life. Like HSt and HX, infantile pyknocytosis can present with hemolytic anemia in infancy. Unlike HSt and HX, infantile pyknocytosis resolves within the first year of life, and the cause is unknown.

MANAGEMENT — Limited data are available on the management of HSt and HX. In general, management is individualized according to the severity of hemolytic anemia.

The value of genetic counseling and testing first-degree relatives is discussed above. (See 'Genetic testing of family members' above.)

Management of hemolysis/hemolytic anemia — Individuals without baseline hemolysis may not require any interventions. However, awareness of the diagnosis may be helpful if they are hospitalized for an acute illness or if they develop an infection that tips the balance between hemolysis and reticulocytosis.

●Folate supplementation – Folate supplementation (typically 1 mg orally per day) is appropriate in individuals with chronic hemolysis.

●Transfusions – Patients with mild or no hemolytic anemia are unlikely to require RBC transfusions. However, transfusions (or exchange transfusions in neonates) may be required in the setting of severe hemolysis. Chronic transfusions are rarely required; the need for chronic transfusions suggests that the original diagnosis may require re-evaluation or that another cause of anemia may have developed in addition to HSt or HX.

Additional information is presented separately according to patient age. (See "Red blood cell (RBC) transfusions in the neonate" and "Red blood cell transfusion in infants and children: Indications" and "Indications and hemoglobin thresholds for RBC transfusion in adults".)

Assessment of iron status — Assessment for iron overload is appropriate in individuals with chronic hemolysis. This is especially important for individuals who have received multiple transfusions; however, iron overload can also occur in the absence of transfusions due to chronic hemolytic anemia. (See 'Clinical manifestations' above.)

Typically, serum iron, ferritin, and transferrin saturation (TSAT; the ratio of iron to transferrin) are measured at the time of diagnosis, with subsequent annual monitoring and testing based on the initial values. Individuals with evidence of iron overload may also have testing for hereditary hemochromatosis (HH) HFE C282Y variant. For patients with HX who are not significantly anemic and who carry a genetic predisposition to iron overload, phlebotomy may be appropriate when ferritin levels are significantly elevated. (See "Approach to the patient with suspected iron overload".)

Gallstones — Patients should be aware of the possibility of pigment gallstones and the typical symptoms.

Evaluation for symptoms is appropriate; however, we do not screen asymptomatic individuals in most cases.

Assessment for and management of gallstones depends on clinical features and severity of the findings. Details are presented in separate topic reviews. (See "Choledocholithiasis: Clinical manifestations, diagnosis, and management" and "Approach to the management of gallstones" and "Gallstone diseases in pregnancy".)

Splenectomy — Splenectomy has been performed in individuals with severe hemolysis. However, there is limited evidence of therapeutic benefit, and the procedure has risks, including those associated with the surgery, risk of infection with encapsulated organisms, and risk of thromboembolism.

Thus, we generally reserve splenectomy as a last resort for patients who are severely symptomatic from splenomegaly. We do not perform splenectomy in the vast majority of patients with HSt or HX. If performed, splenectomy should be undertaken with extreme caution, especially in individuals carrying a PIEZO1 variant, as thrombotic complications following splenectomy have been mostly described in individuals with PIEZO1 variants [137].

Individuals with HSt or HX who undergo splenectomy have a much greater risk of vascular complications including thromboembolic events and/or vaso-occlusive episodes [65,117,124]. Several case reports describe development of pulmonary hypertension due to multiple thromboemboli following splenectomy in HSt [65,117]. (See 'Other findings' above.)

Additional pre-splenectomy considerations are presented separately. (See "Elective (diagnostic or therapeutic) splenectomy" and "Prevention of infection in patients with impaired splenic function".)

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

SUMMARY AND RECOMMENDATIONS

●Definitions – Stomatocytes are red blood cells (RBCs) with a mouth-shaped area of central pallor (picture 1) caused by conversion of the biconcave disc to a uniconcave cup (picture 2). Xerocytes are dense, hyperchromic RBCs with an elevated mean cell hemoglobin concentration (MCHC) (picture 3). Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX) have been classified according to RBC appearance and the degree of cell hydration. (See 'Definitions and classification' above.)

●Genetics – HSt and HX are typically autosomal dominant. Some cases of HSt and HX are caused by pathogenic variants in the PIEZO1 gene, which encodes a mechanosensitive ion channel. Some cases of dehydrated HSt are caused by pathogenic variants in KCNN4, which encodes the Gardos channel. Other implicated genes include RHAG and SCL4A1/BAND3/AE1. (See 'Genetics' above.)

●Disease mechanisms – Several ion transport systems continuously regulate RBC water, cation, and anion content (figure 1). Stomatocytes and xerocytes form when changes in RBC cation content cause compensatory changes in cell water (figure 2). The resulting decrease in deformability of the RBCs predisposes them to hemolysis. The table summarizes other disorders of RBC volume (table 1). (See 'Pathophysiology' above.)

●Typical findings – HSt and HX cause variable compensated hemolysis and hemolytic anemia, with abnormal RBC morphologies. Iron overload can occur, even without transfusions. Some individuals develop splenomegaly and/or pigment gallstones. Some have an increased risk of vascular and/or thromboembolic complications. (See 'Clinical manifestations' above.)

●Diagnostic testing – The diagnosis may be suspected due to a positive family history, unexplained non-immune hemolysis, unexplained elevated MCHC and mean corpuscular hemoglobin (MCH), or stomatocytes or xerocytes on the blood smear. Testing proceeds sequentially. All individuals should have a review of the complete blood count (CBC), RBC indices, and blood smear, and testing for hemolysis, including a direct antiglobulin test (DAT; Coombs test). For those with suggestive findings, additional testing includes osmotic fragility, ektacytometry, and/or genetic testing. Resources for obtaining this testing are listed above. (See 'Evaluation' above and 'Diagnosis' above.)

●Differential diagnosis – The differential diagnosis includes other inherited hemolytic anemias (figure 4), other inherited causes of stomatocytosis such as Southeast Asian ovalocytosis, acquired causes of stomatocytosis (liver disease, alcohol, medications), and infantile pyknocytosis. An approach to determining the cause of stomatocytosis or a high MCHC is illustrated in the algorithm (algorithm 1). (See 'Differential diagnosis' above.)

●Management – Management is individualized according to the severity of hemolysis. Those with chronic hemolysis are given folic acid. Transfusions may be needed intermittently. Monitoring for iron overload is appropriate. Symptomatic gallstones should be evaluated and treated. (See 'Management of hemolysis/hemolytic anemia' above and 'Assessment of iron status' above and 'Gallstones' above.)

●Role of splenectomy – Evidence for improvement with splenectomy is limited, and severe complications including thromboembolic events and pulmonary hypertension have been reported, especially in individuals with pathogenic variants in PIEZO1. Postoperative infection and surgical complications can occur. We generally reserve splenectomy for patients with severe hemolysis or severe symptoms related to splenomegaly; for those with PIEZO1 variants, we recommend not performing splenectomy (Grade 1C). (See 'Splenectomy' above.)

ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges extensive contributions of Donald H Mahoney, Jr, MD, to earlier versions of this topic review.