Unstable hemoglobin variants

INTRODUCTION — Some mutations of globin genes decrease the stability and solubility of the hemoglobin molecule in the red blood cell (RBC). Substitutions in the primary sequence of globin in these unstable hemoglobins can alter the tertiary or quaternary structure of the molecule and result in a globin polypeptide/hemoglobin tetramer that is unstable and precipitates intracellularly. These intra-erythrocytic precipitates are detectable by supravital staining as dark globular aggregates called Heinz bodies. The affected RBCs have a reduced life span, producing a hemolytic disorder of varied severity called the congenital Heinz body hemolytic anemia syndrome. (See 'Blood smear and Heinz body preparation' below.)

This topic reviews the evaluation and management of unstable hemoglobins (hemoglobin mutations associated with the Heinz body hemolytic anemia syndrome).

Separate topic reviews discuss other causes of hemolytic anemia associated with Heinz body formation and general approaches to the evaluation of hemolytic anemia:

●G6PD deficiency – (See "Diagnosis and management of glucose-6-phosphate dehydrogenase (G6PD) deficiency".)

●Thalassemia – (See "Diagnosis of thalassemia (adults and children)".)

●Hemolytic anemia in children – (See "Approach to the child with anemia" and "Overview of hemolytic anemias in children".)

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

The structure and function of normal hemoglobins are also discussed separately. (See "Structure and function of normal hemoglobins".)

EPIDEMIOLOGY — Unstable hemoglobin variants are rare, inherited mutations affecting globin genes. Thus, with a few exceptions, each variant is generally limited to a single pedigree [1].

Exceptions include the following mutations, which are seen more commonly:

●Hemoglobin Köln (HBB val98met), which has been detected in several different pedigrees and geographic locations [2]. (See 'Hemoglobin Köln' below.)

●Hemoglobin Hasharon (HBA2 asp47his), which is found predominantly among individuals with Ashkenazi Jewish ancestry, causing hemolysis in newborns [3].

●Hemoglobin Zürich (HBB his63arg), which has been detected in several different pedigrees and geographic locations and is susceptible to oxidant drug-induced hemolysis. (See 'Hemoglobin Zürich' below.)

GENETIC VARIANTS AND THEIR EFFECT ON PROTEIN STRUCTURE

Overview of genetics — Specific variants discussed herein are referred to using the classical amino acid numbering system related to the mature globin peptide, not the Human Genome Variation Society (HGVS) numbering system. (See "Basic genetics concepts: DNA regulation and gene expression", section on 'Sequence variants'.)

Of the over 1200 hemoglobin variants (mutations) that have been described, there are approximately 150 that are classified as unstable alpha globin or beta globin variants on the online Globin Gene Server (http://globin.cse.psu.edu/).

Mutations conferring instability can also affect the gamma globin and delta globin chains. Delta globin chain instability does not have a clinical phenotype because the delta globin-containing hemoglobin (Hb A2) is present at very low concentration. Unstable gamma globin variants can cause neonatal hemolytic anemia that resolves as fetal hemoglobin (Hb F) is replaced by adult hemoglobin in the first months of life. In addition to instability, many of these variants also have increased or decreased oxygen affinity as the mutations may interfere with the transitions of the hemoglobin tetramer between oxygenated (R) to deoxygenated (T) states, in addition to causing instability. Auto-oxidation and methemoglobinemia are also common features. In the discussion below, the affected globin chain is designated by the gene name followed by the amino acid substitution. As an example, HBB ala138pro refers to an alanine to proline substitution at position 138 in the mature beta globin chain.

The mechanisms that contribute to hemoglobin instability vary, and these are outlined below. (See 'Altered globin folding/assembly' below and 'Altered heme-globin interactions' below and 'Hyperunstable variants' below.)

The inheritance of the majority of unstable hemoglobins is autosomal dominant (figure 1), and affected individuals are heterozygous for the mutation; however, homozygosity may rarely also occur, especially for the more common variants or in populations with a high degree of consanguinity [4,5].

Altered globin folding/assembly — Some mutations causing unstable hemoglobins affect globin folding or assembly. This may be at the level of the secondary structure (initial folding of the protein into an alpha helix), tertiary structure (overall three-dimensional shape of the globin chain), or quaternary structure (interaction between globin subunits) of the hemoglobin molecule. As examples:

●Approximately 75 percent of the hemoglobin protein is in the form of an alpha helix, and disruption of this secondary structure can reduce the solubility of the globin subunit. Unstable hemoglobin variants can result from the introduction of proline in the helical structure, the substitution of glycine by a mutation in invariant positions in the interhelical bends, and/or by amino acid insertions and deletions.

●At least 33 unstable hemoglobin variants involve the introduction of a proline residue. Proline residues disrupt the alpha helix of the globin chain, except when present at the ends of the helical section. Alpha helical regions of the globin subunits must be folded so that the hemoglobin tetramer can assume its tight globular configuration. An example of a variant that disrupts this folding is hemoglobin Brockton (HBB ala138pro), an abnormality that is associated with mild anemia. Crystallographic examination showed that the tertiary structure was disrupted in the vicinity of the mutated residue [6].

●Substitutions in the amino acid residues that point inward can occur with no change in stability/solubility as long as the incoming residue is not charged, is not bulky, or there is no loss of critical nonpolar residues that prevent water from entering the interior of the molecule.

Introduction of charged residues in the interior of the hemoglobin molecule or loss of inter-subunit contact hydrogen bonds or salt bridges in the alpha1beta1 contact area makes the protein unstable. This area of contact is normally very strong and difficult to dissociate. When a substitution weakens the contact area, it promotes dissociation of the alpha1beta1 dimers into monomers, loosening their heme-globin interaction and favoring methemoglobin formation. (See 'Altered heme-globin interactions' below.)

Altered heme-globin interactions — Heme binding to globin is important for the oxygen binding properties and the stability and solubility of the molecule. Several of the mutations involving residues with defined interactions with the heme groups that result in protein instability are depicted in the figure (figure 2). These include substitutions that introduce a charged side chain into the heme pocket where a nonpolar side chain existed previously, deletions involving residues that directly interact with heme, and non-tyrosine substitutions of the proximal histidine [7]. Observations in individuals with hemoglobin Köln and hemoglobin Zürich illustrate these mechanisms.

Hemoglobin Köln — Hemoglobin Köln is the most frequently diagnosed unstable hemoglobin in the Western world. It is characterized by mild anemia, reticulocytosis, splenomegaly, and pigmenturia; it comprises 10 to 25 percent of the total hemoglobin in heterozygotes [2]. The presence of the hemoglobin Köln mutation at a CpG dinucleotide leads to deamidation of cytosine to thymine, perhaps accounting for its high incidence compared with other unstable variants. This mutation involves a residue in the heme pocket (FG5) that is very close to the proximal histidine (F8His) that is involved in a valency bond with heme iron.

The functional properties of hemoglobin Köln include a high oxygen affinity in the absence of compensatory erythrocytosis and easy heme dissociation. Of special interest are the abnormalities in the membrane of erythrocytes containing hemoglobin Köln. When the affected individual's red blood cells (RBCs) are exposed to hydrogen peroxide stress, they produce twice the malonylaldehyde (a lipid peroxidation product) than normal RBCs. Two sorts of abnormal membrane aggregates have been found in individuals with hemoglobin Köln who have undergone splenectomy:

●A disulfide bond between membrane band 3 (also known as anion exchanger [AE]-1) and globin.

●Aggregates of spectrin and other membrane components, which have an amino acid composition similar to normal membranes exposed to malonylaldehyde.

Membrane-associated aggregates are thought to reduce the deformability of hemoglobin Köln erythrocytes, thereby contributing to the hemolysis. In addition, the denatured hemoglobin protein co-clusters with the RBC membrane band 3, resulting in increased binding of autoantibodies against band 3 [8]. This process may be important physiologically in aged RBCs when hemoglobin begins to denature; it promotes recognition by macrophages and contributes to the removal of senescent cells.

Red cells containing hemoglobin Köln have a green fluorescent pigment due to dipyrroles, the same pigment found in the pigmented urine of these individuals [9]. The partial lack of heme molecules in some of the tetramers of hemoglobin Köln makes the use of pulse oximetry unreliable in these and some other individuals with unstable hemoglobin variants [10]. (See 'Other manifestations' below.)

Hemoglobin Zürich — In hemoglobin Zürich (HBB his63arg), substitution of the distal histidine in the beta globin chain enlarges the space available for ligand binding around the iron, fundamentally modifying its ligand properties [11,12]. This steric change has little effect on the binding of oxygen to iron, but it increases the affinity for carbon monoxide (CO). Carboxyhemoglobin levels are very high in smokers with hemoglobin Zürich due to the CO content of tobacco smoke, but they are also elevated in nonsmokers who carry the mutation due to endogenous CO production from heme catabolism. CO stabilizes the hemoglobin tetramer, and this partially protective effect of carboxyhemoglobin Zürich reduces the rate of hemolysis and the frequency of Heinz body formation, especially among smokers with the hemoglobin Zürich mutation [12,13].

Individuals heterozygous for hemoglobin Zürich usually have normal hemoglobin levels with little hemolysis, but they have a special susceptibility to hemolytic crisis induced by oxidant agents like sulfanilamide. This is because the increased size of the heme pocket in the hemoglobin of these individuals allows oxidant agents to enter the heme pockets and directly oxidize heme, producing methemoglobin at levels reported to be as high as 40 percent. As noted above, smoking is protective due to stabilization of the tetramer by CO.

Hyperunstable variants — Some hemoglobins are so unstable that they are either barely detectable or undetectable in the hemolysate. These rare hemoglobins are called hyperunstable hemoglobin variants; they have also been called thalassemic hemoglobinopathies, as the rapid destruction of the variant globin chain causes unbalanced globin synthesis, the hallmark of thalassemia. (See "Pathophysiology of thalassemia", section on 'Globin chain imbalance'.)

Hyperunstable variants presumably are synthesized normally but are destroyed rapidly after synthesis because of extreme instability, creating the phenotype of a dominantly inherited thalassemia. Individuals with these variants are heterozygous for the mutated globin but have a phenotype of severe thalassemia.

Examples of hyperunstable variants include the following:

●Hemoglobin Indianapolis – Hemoglobin Indianapolis (HBB leu106arg, previously identified as cys112arg) was one of the first examples of a hyperunstable hemoglobin phenotype to be described [14,15]. Hemoglobin Indianapolis was not detectable in the hemolysate, but its beta globin chain could be detected by biosynthetic means and had a calculated half-life of less than 10 minutes. The proband and his daughter were transfusion-dependent with all the features of severe beta thalassemia. Unlike the typical severe thalassemia, the phenotype was inherited in a dominant manner. Erythrocyte membrane damage by the labile beta globin chain and unpaired alpha-globin chains were thought to account for the severe phenotype.

●Hemoglobin Petah Tikva – Hemoglobin Petah Tikva (HBA1 or HBA2 ala110asp) was found in two unrelated Iraqi Jewish children, the phenotype appeared to be an alpha thalassemia with hematologic characteristics of mild hemoglobin H disease (see "Diagnosis of thalassemia (adults and children)", section on 'Anemia'), suggesting that the children had inherited only a single normal alpha chain gene instead of the usual four [16]. The hemoglobin variant could not be separated by electrophoresis, but the abnormal alpha chain was detected by high performance liquid chromatography (HPLC). The hemolysate was unstable, and the precipitated material was enriched in the mutant globin chain. Each child had one parent with alpha thalassemia trait and one parent who was heterozygous for hemoglobin Petah Tikva. Each affected individual appeared to be compound heterozygotes for both alpha thalassemia trait and hemoglobin Petah Tikva.

Synthesis studies in heterozygotes for hemoglobin Petah Tikva revealed that only traces of the Petah Tikva alpha globin were synthesized by reticulocytes. In the compound heterozygotes, both mutated and normal alpha chains were synthesized by bone marrow erythroid precursors at rates related to their concentration in blood. Thus, the phenotype was the consequence of the triad of alpha thalassemia, the unstable Petah Tikva alpha globin variant, and its premature termination of synthesis during erythroid differentiation.

●Hemoglobin Bristol-Alesha – Hemoglobin Bristol-Alesha is caused by a GTG to ATG mutation at codon 67 (classical numbering) in HBB, resulting in a change from val67met that is followed by post-translational oxidation of the encoded methionine and subsequent conversion to aspartic acid [17,18]. Four cases of this variant have been described. All of the affected individuals had hemolytic anemia and splenomegaly, and all received transfusions. In one case, hemolytic anemia was enhanced following splenectomy at four years of age. Splenectomy caused accumulation of abnormal beta globin chains with an increased proportion of beta67met. It was speculated that the high fraction of the beta67met molecule caused the extreme hemoglobin instability, resulting in a thalassemic hyperunstable hemoglobinopathy and very severe clinical findings.

●Hemoglobin Toms River – Hemoglobin F Toms River (HBG2 val67met-asp) is an unstable Hb F variant with some features similar to hemoglobin Bristol-Alesha, but the abnormality occurs in the G gamma globin gene. It was first identified in a neonate with cyanosis and anemia; her father also had neonatal cyanosis and anemia, and as an adult he had well-compensated hemolytic anemia with a hemoglobin level of 11 g/dL and reticulocytosis (20 percent) [19]. Her father did not have measurable Hb F so the cause of hemolysis was unclear. It was speculated that the encoded mutation to methionine was responsible for cyanosis, and the post-translational oxidative conversion to aspartate caused instability. The phenotype was not as severe as that of hemoglobin Bristol-Alesha, likely due to the lower concentration of hemoglobin F Toms River in erythrocytes.

PATHOPHYSIOLOGY OF HEMOLYSIS

Hemoglobin precipitation and Heinz body formation — Denaturation of hemoglobin leads to its precipitation in the RBC. As a general rule, weakening of the heme-globin bond will accelerate hemoglobin denaturation.

Heinz bodies are intraerythrocytic hemoglobin precipitates that form when hemoglobin denatures and produces hemichromes. Hemichromes are molecules generated when the heme dissociates from the heme pocket and rebinds elsewhere in the globin molecule after the alpha or beta chains have undergone some degree of uncoiling or denaturation. Formation of irreversible hemichromes appears to be an indispensable stage in the formation of Heinz bodies [20,21].

Heinz bodies adhere at least partially to the cytosolic (internal) surface of the RBC membrane by hydrophobic interactions [22]. Band 3, one of the most abundant erythrocyte transmembrane proteins, has a highly glycosylated portion on the external surface of the RBC, a hydrophobic portion spanning the lipid bilayer, and an N-terminal cytosolic domain that has two conformational independent portions separated by a protease sensitive hinge region. The N-terminal domain is highly negatively charged and has a high content of proline and other amino acid residues that do not accommodate well in an alpha helix. Hemoglobin and hemichromes bind the most N-terminal portion of the band 3 cytosolic domain [23,24]. The band 3 cytosolic domain can thread into the positively charged central cavity of hemoglobin, which is why this domain preferentially and reversibly binds deoxyhemoglobin [24].

Heinz bodies generally are cleared by splenic macrophages as part of their normal reticuloendothelial function. This provides a possible explanation for the absence of Heinz bodies in some individuals with unstable hemoglobin variants. (See 'Blood smear and Heinz body preparation' below.)

Hemolysis — Not all of the unstable hemoglobin variants are sufficiently abnormal to result in hemolytic anemia. As a result, not all affected individuals will have hemolysis; in those who do have hemolysis, it may range from mild to severe. Hemolysis is typically both intravascular and extravascular.

The mechanisms are several-fold:

●Formation of Heinz bodies may lead to reduced deformability, which in turn may cause RBCs to be trapped preferentially in the spleen.

●Heinz bodies may also cause adjacent portions of the RBC membrane to be excised in the spleen (a process known as "pitting"), leading to the formation of "bite" or "blister" cells [25]. Cycles of membrane loss convert RBCs into spherocytes, which are more rigid than normal RBCs and hence have a shorter lifespan.

●Free heme that becomes liberated from an unstable hemoglobin may cause heme iron to become decompartmentalized, in turn damaging the RBC membrane and RBC proteins, including hemoglobin. This may result from several intermediates, including peroxidized membrane components, generation of free radicals, methemoglobin formation, and hemoglobin denaturation.

Pigmenturia — Heme is ferrous protoporphyrin IX, which consists of four pyrrole rings linked by methenyl bridges. Heme oxygenase cleaves heme at the alpha-methene (methenyl) bridges to form biliverdin.

Pigmenturia is a color change in the urine due to the accumulation of fluorescent compounds in the plasma. These are thought to be dipyrrole methenes of the mesobilifuscin group. The origin of these fluorescent compounds is not clear. Their structure, two pyrrole rings still bound to each other by a methenyl bridge, suggests the malfunctioning of the methenyl oxygenase enzyme.

Pigmenturia is different from hemoglobin in the urine due to intravascular hemolysis (hemoglobinuria). Inherited unstable hemoglobin variants that cause Heinz body hemolytic anemia typically do not cause hemoglobinuria.

Fluorescent dipyrroles are also present in Heinz bodies [9]. (See 'Other manifestations' below.)

CLINICAL MANIFESTATIONS — The typical clinical manifestations of an unstable hemoglobin variant include varying degrees of hemolysis, hemolytic anemia, and complications of hemolysis [2,3]. Individuals with congenital nonspherocytic hemolytic anemia that is unexplained by more common causes such as RBC enzymopathies, immune-related hemolysis, thalassemia, or sickle cell disease should be evaluated for an unstable hemoglobin.

Presentation — Individuals with unstable hemoglobins can present in a number of ways:

●Congenital nonspherocytic hemolytic anemia with splenomegaly and pigmented (bilirubin) gallstones

●Heinz body hemolytic anemia with sensitivity to oxidant drugs such as sulfonamides

●Mild or minimal anemia with reticulocytosis out of proportion to the hemoglobin level

●In cases of the hyperunstable variants, a thalassemia-like peripheral blood picture with hypochromic RBCs

●Increased formation of methemoglobin

Many unstable variants present during childhood. Those affecting the gamma globin chains present neonatally. Some individuals with mild, compensated hemolysis will not come to medical attention until later in life or if they have a precipitating event such as a severe infection or oxidant drug exposure. Others may have unexplained chronic mild anemia but are diagnosed only as adults when the possibility of an unstable hemoglobin is evaluated.

Clinical variability — Sources of variability in presentation include which globin chain is affected, whether the mutation alters the oxygen affinity of the hemoglobin, environmental factors such as smoking and medications, and other unidentified genetic or environmental modifiers.

●Globin chain affected – Individuals with unstable variants affecting the gamma globin chain have fetal and neonatal hemolysis that resolves during the first several months of life as hemoglobin synthesis shifts from fetal hemoglobin (Hb F) to adult hemoglobin A (Hb A) (ie, from alpha2gamma2 to alpha2beta2). (See "Fetal hemoglobin (Hb F) in health and disease", section on 'Biology of Hb F'.)

This is seen with the gamma chain variant hemoglobin Poole (HBG2 trp130gly) [26].

By contrast, individuals with unstable beta globin variants may appear at birth not to have a hemolytic anemia and may develop progressive hemolysis during the first year of life as beta globin chain production increases. (See "Structure and function of normal hemoglobins".)

●Oxygen affinity – The degree of anemia associated with an unstable hemoglobin variant may be affected by the oxygen affinity of the hemoglobin produced, which in turn can shift the hemoglobin-oxygen dissociation curve and lead to more or less erythropoietin production. As an example, some unstable variants with high oxygen affinity have milder anemia because they promote erythropoietin production and increased RBC production [1].

●Environmental factors – A number of environmental factors may affect the degree of hemolysis and/or anemia.

•Smoking – Smoking may cause increased carbon monoxide levels, which protects against hemoglobin oxidization in some of the unstable variants, especially hemoglobin Zürich. (See 'Hemoglobin Zürich' above.)

•Medications – Medications with oxidant properties (eg, sulfonamides) may precipitate hemolytic episodes with some of the unstable variants [27]. Hemolytic crisis upon exposure to oxidant drugs has been seen with hemoglobin Zürich, hemoglobin Hasharon, hemoglobin Shepherds Bush, and hemoglobin Peterborough. These crises are generally self-limited. Implicated medications are similar to those that cause hemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. However, the association is poorly documented in many cases. (See "Drug-induced hemolytic anemia", section on 'Oxidant injury'.)

•Infections – Viral or bacterial infections may precipitate hemolytic episodes with some of the unstable variants. The mechanism of infection-mediated hemolysis is not clear, but pyrexia and transient acidosis may contribute since both can increase the denaturation of the hemoglobin protein.

•Aplastic crisis – Infection with parvovirus B19 may precipitate a transient aplastic crisis due to toxicity to RBC precursors in the bone marrow in the setting of chronic hemolytic anemia. (See "Clinical manifestations and diagnosis of parvovirus B19 infection", section on 'Transient aplastic crisis'.)

●Other modifiers – The presence of other modifiers is inferred from the dramatic variations in clinical severity within the same pedigree or among different pedigrees. As an example, the disease manifestations vary widely in adults with hemoglobin Hasharon and hemoglobin Zürich, from mild hemolytic anemia to no hemolysis at all. The presence of an unstable beta globin variant as a compound heterozygote with a beta thalassemia mutation will increase the concentration of the unstable variant, making hemolysis more readily detectable. Similarly, levels of unstable alpha globin variants will be higher in the presence of alpha thalassemia. Some variants interact abnormally with alpha hemoglobin stabilizing protein (AHSP), which causes their instability [28]. Epistatic effects of other non-linked modifier genes might also be responsible for these differences in carriers of the same globin-gene mutation.

Hemolytic anemia — Hemolytic anemia is the chief clinical feature of unstable hemoglobins. The intensity of hemolysis may vary considerably depending on the specific mutation [27]. Some individuals may have chronic hemolysis, while others may have an abrupt increase in hemolysis over steady state levels following an acute infection or exposure to oxidant medications. (See 'Clinical variability' above.)

As with other forms of hemolytic anemia, the reticulocyte count is elevated unless there is a concomitant vitamin or mineral deficiency that impairs hematopoiesis (table 1). The haptoglobin is often low, the lactate dehydrogenase (LDH) may be increased, and the direct antiglobulin (Coombs) test (DAT) is negative because there is no immune component. The white blood cell (WBC) count and platelet count are usually normal but may be increased. (See "Approach to the child with anemia", section on 'Laboratory evaluation' and "Diagnostic approach to anemia in adults", section on 'Evaluation based on CBC/retic count'.)

The peripheral blood smear may show a variety of abnormal RBC morphologies including bite cells and/or spherocytes. The RBC indices may show a normocytic anemia with an increased red cell distribution width (RDW) due to the presence of small spherocytes and large reticulocytes. Heinz bodies are not seen in the peripheral blood smear and can only be visualized using a supravital stain. (See 'Blood smear and Heinz body preparation' below.)

Other manifestations — Chronic hemolysis may be associated with effects on other organs including the following [27,29]:

●Gallstones – Pigmented (bilirubin) gallstones may form in the setting of chronic hemolysis.

●Splenomegaly – Splenomegaly has been reported with some unstable hemoglobin variants; hemoglobin Köln is the most commonly reported. The clinical presentation is of a slight to moderate, usually normocytic anemia that may be accompanied by splenomegaly.

●Pigmenturia – Some unstable hemoglobin variants produce pigmenturia, the severity of which is unrelated to the severity of hemolysis. As examples, pigmenturia can be seen in individuals with hemoglobin Köln who have severe hemolysis and in individuals with hemoglobin Zürich who have very mild hemolysis.

●Methemoglobinemia – Some unstable hemoglobin variants may cause methemoglobinemia (oxidation of heme iron from the ferrous [Fe²⁺] to the ferric [Fe³⁺] state, which renders it unable to bind O₂). This may cause symptoms of hypoxemia out of proportion to the degree (if any) of anemia, and the blood may appear dark red or chocolate colored. At low levels of methemoglobinemia (eg, ≤20 percent), oxygen saturation may appear normal by pulse oximetry. At higher levels of methemoglobinemia (eg, >20 percent), oxygen saturation by pulse oximetry may be low but will still underestimate the degree of true oxygen desaturation. (See "Methemoglobinemia".)

●Spuriously low hemoglobin oxygen saturation by pulse oximetry reading – An unexpectedly low hemoglobin oxygen saturation by pulse oximetry can be caused by certain unstable hemoglobins such as hemoglobin Köln. Some variants have been reported to interfere with pulse oximetry readings and produce a falsely low SpO₂ [10]. For most of these, the arterial blood gas will give a normal SaO₂ reading. An exception is hemoglobins that have intrinsically low oxygen affinity, for which pulse oximetry will give an accurately low SaO₂. Variants with low oxygen affinity may have normal or even increased tissue oxygen delivery, and the benefit of their identification is in avoiding other extensive evaluations.

●Less common complications related to NO depletion – Several other complications may occur due to depletion of nitric oxide (NO); these are less common than the manifestations listed above. Examples include pulmonary hypertension, priapism, and thrombosis [30].

●Moyamoya – Steno-occlusive cerebral arteriopathy has been reported in several individuals with several unstable hemoglobins, including:

•Hemoglobin Alesha [31]

•Hemoglobin Fairfax [32]

•Hemoglobin Southampton (Casper) [33,34]

The pathophysiology is unclear, but it likely involves endothelial activation, injury, and dysfunction driven by chronic intravascular hemolysis. (See "Moyamoya disease and moyamoya syndrome: Etiology, clinical features, and diagnosis".)

Splenectomy may be a risk factor for moyamoya, at least in hemoglobin Southampton [33,34]. (See 'Splenectomy' below.)

DIAGNOSTIC EVALUATION

Clinical evaluation — The diagnosis of an unstable hemoglobin variant is suspected in a child or adult with unexplained hemolytic anemia. Most present in childhood or adolescence, but many are diagnosed as adults because chronic mild hemolytic anemia was not appreciated or was attributed to another condition [35-37].

●History – The history may reveal a personal or family history of unexplained anemia, gallstone disease, or hyperbilirubinemia. Some individuals or family members may have had anemia severe enough to require transfusion, but this is not common.

●Examination – The physical examination may show pallor, jaundice, or splenomegaly but often is unremarkable.

●Laboratory testing – Initial laboratory testing includes a complete blood count (CBC) with reticulocyte count to determine whether other blood cells are affected and whether the anemia is likely to be caused by hemolysis or by decreased red blood cell (RBC) production. In individuals with an unstable hemoglobin variant, the other cell lines are unaffected and the reticulocyte count is increased.

Additional CBC findings consistent with an unstable hemoglobin variant include normocytic anemia and increased red cell distribution width (RDW), especially if there is a reticulocytosis.

Individuals may also have other findings of hemolytic anemia on routine evaluations, including increased bilirubin, increased lactate dehydrogenase (LDH), decreased haptoglobin, and pigmenturia, depending on the severity of intravascular hemolysis. Tests for autoimmune hemolysis such as the direct antiglobulin test (DAT, direct Coombs) typically are negative. (See "Overview of hemolytic anemias in children" and "Diagnosis of hemolytic anemia in adults".)

Rarely, it may be useful to obtain baseline pulse oximetry, in case the unstable hemoglobin is one that produces a falsely low reading [10]. This may be helpful for preventing extensive evaluations for hypoxemia.

Blood smear and Heinz body preparation — Review of the peripheral blood smear is an important component of any hemolytic anemia evaluation. This is important both to determine if findings are consistent with an unstable hemoglobin as well as to eliminate other potential causes of hemolytic anemia, some of which may be life threatening and/or require more urgent interventions. (See "Approach to the child with anemia", section on 'Blood smear' and "Diagnostic approach to anemia in adults", section on 'Hemolysis'.)

Typical findings on the blood smear in individuals with an unstable hemoglobin may include anisocytosis, prominent basophilic stippling, and/or microspherocytes. The RBCs may be hypochromic leading to a thalassemia-like picture, especially with the hyperunstable variants (see 'Hyperunstable variants' above). Howell-Jolly bodies may be seen, especially in individuals who have had a splenectomy, and nucleated RBCs may be present, especially if hemolysis is severe. Importantly, these abnormalities on the peripheral blood smear are not specific for unstable hemoglobins and may be seen with various other conditions associated with hemolysis. (See 'Differential diagnosis' below.)

A Heinz body stain, if available, may be helpful for cases of suspected unstable hemoglobin variants, provided that the laboratory has the expertise to interpret the results correctly. Heinz bodies are RBC inclusions caused by denatured hemoglobin proteins (see 'Hemoglobin precipitation and Heinz body formation' above). Their presence is consistent with a diagnosis of an unstable hemoglobin but not required [38]. Detection of Heinz bodies requires incubation of RBCs with a supravital stain such as methylene blue, crystal violet, or brilliant cresyl blue. Heinz bodies appear as single or multiple pale purple inclusions that are 2 microns in diameter or less; they frequently appear to be attached to the cell membrane. The test is inexpensive and can be performed rapidly, but many laboratories do not have experience with evaluation and interpretation. Heinz bodies may be found in freshly drawn samples of peripheral blood but, in most cases, incubation for 24 hours in the absence of glucose is required for them to form. Due to the extreme variability inherent in this test, a normal blood sample as a control should always be run simultaneously. Heinz bodies become easier to detect after splenectomy.

Specialized testing

Hemoglobin stability testing — Heat stability (also called heat denaturation) or isopropanol stability testing are simple screening methods that, if positive, suggest the presence of an unstable hemoglobin variant. An unstable hemoglobin is suggested if a precipitate forms at high temperature or in isopropanol [21,39-42]. A positive test should be followed by genetic testing to define the mutation and confirm the diagnosis. (See 'Genetic testing' below.)

Genetic testing — For individuals with unexplained hemolytic anemia for whom other screening tests do not reveal a diagnosis and/or an unstable hemoglobin is suspected, genetic testing for unstable hemoglobin variants should be pursued. Genetic testing is the most definitive means of establishing the presence of a pathogenic variant in a globin gene. Interpretation is most reliable for variants that have already been described.

Genetic testing is performed by DNA sequence analysis using leukocytes from peripheral blood [35]. Testing may be ordered from some commercial and academic laboratories, such as the following:

●Boston University Hemoglobin Diagnostic Reference Laboratory [43]

●Cincinnati Children's Hospital Medical Center Erythrocyte Diagnostic Laboratory [44]

●Mayo Medical Laboratories [45]

●Quest Diagnostics [46]

Hemoglobin analysis (separation) — Additional tests that may be performed and may reveal an abnormal hemoglobin protein include high performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), or, less commonly, gel-based hemoglobin electrophoresis. In some cases, these tests may be performed to evaluate the possibility of another hemoglobinopathy such as thalassemia.

●HPLC and CZE – These are widely used methods for separating hemoglobins and measuring their relative percentages in the hemolysate. Approximately 25 percent of unstable hemoglobins cannot be detected by HPLC, CZE, or other means of hemoglobin separation; thus, a negative test in the setting of a consistent clinical picture does not eliminate the possibility of an unstable hemoglobin [27].

●Hemoglobin electrophoresis – This uses a gel method to separate hemoglobin proteins based on their size and charge, although this approach is much less commonly used and is not very sensitive for unstable hemoglobins.

The utility of these protein-based tests for evaluating unstable hemoglobin variants has been supplanted by the availability of genetic testing when clinical evaluation suggests the possibility of these rare hemoglobinopathies. If this testing has been performed and suggests the possibility of an unstable hemoglobin, the ultimate diagnosis should be pursued using genetic testing as described above. (See 'Genetic testing' above.)

Additional information about these tests is presented in more detail separately. (See "Methods for hemoglobin analysis and hemoglobinopathy testing".)

Diagnosis — The diagnosis of an unstable hemoglobin variant is established in an individual with chronic hemolytic anemia by documentation of a causative globin gene defect by genetic testing or, less reliably, by identification of a hemoglobin variant by HPLC or CZE in the appropriate clinical setting (findings on CBC and peripheral blood smear, known affected family member).

We favor genetic testing because it is widely available and provides definitive genetic evidence of the variant. Many laboratories do not perform hemoglobin HPLC or CZE routinely. For settings in which HPLC or CZE is available, genetic testing will often be required regardless of the result (ie, to make the diagnosis if HPLC fails to find a variant; to confirm the diagnosis if HPLC identifies a variant phenotypically) [47,48]. However, in selected cases, the clinical picture along with HPLC or CZE by a lab with expertise in unstable hemoglobins may be used to establish the diagnosis.

For family members of an individual with a variant that has been confirmed by genetic testing, diagnosis may be made by the clinical picture along with HPLC or CZE.

DIFFERENTIAL DIAGNOSIS — The differential diagnosis of an unstable hemoglobin variant includes other causes of inherited and acquired hemolytic anemia, especially those associated with Heinz bodies [37]:

●Red blood cell (RBC) enzymatic defects – A number of inherited RBC enzymatic defects cause non-immune hemolytic anemia. These include deficiencies in pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase, glutathione synthase, and gamma-glutamyl cysteine synthetase. Like unstable hemoglobin variants, these enzymatic defects may present with varying degrees of chronic non-immune hemolysis with or without exacerbations during infection or upon exposure to certain medications; Heinz bodies and/or bite cells may be present. Unlike unstable hemoglobin variants, laboratory testing in these enzymatic disorders reveals the specific enzymatic deficiency. (See "Overview of hemolytic anemias in children", section on 'Intrinsic hemolytic anemias' and "Diagnosis of hemolytic anemia in adults", section on 'Conceptual framework'.)

●RBC membrane/cytoskeletal defects – A number of inherited defects in the RBC membrane and/or cytoskeleton can cause non-immune hemolytic anemia. These include spherocytosis, elliptocytosis, stomatocytosis, and xerocytosis. Like unstable hemoglobins, these membrane/cytoskeletal defects cause chronic non-immune hemolysis with abnormal RBC morphologies on the blood smear. Unlike unstable hemoglobins, these other defects are associated with a more uniform population of cells with a specific shape, and with abnormal laboratory testing such as osmotic gradient ektacytometry, eosin-5 maleimide binding, and osmotic fragility. (See "Hereditary spherocytosis" and "Hereditary elliptocytosis and related disorders" and "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)".)

●Other hemoglobinopathies – Other inherited hemoglobinopathies include the thalassemias, sickle cell disease (SCD), hemoglobin C (Hb C) disease, and others; many of these are much more common than the unstable hemoglobins. Like the unstable hemoglobins, these hemoglobinopathies can be associated with chronic non-immune hemolysis and abnormal-appearing RBCs on the blood smear. Unlike these other hemoglobinopathies, the unstable hemoglobins have laboratory evidence of the unstable variant (eg, from review of the blood smear, RBC indices, and HPLC or CZE). (See "Methods for hemoglobin analysis and hemoglobinopathy testing" and "Diagnosis of sickle cell disorders".)

●Autoimmune hemolytic anemia (AIHA) – AIHA is one of the more common causes of acquired hemolytic anemia, both in children and adults. Like unstable hemoglobin variants, individuals with AIHA may have varying degrees of hemolytic anemia. Unlike unstable hemoglobin variants, individuals with AIHA typically have positive testing for antibodies on RBCs (ie, they have a positive direct antiglobulin [Coombs] test [DAT] or indirect antiglobulin [Coombs] test [IAT]). (See "Autoimmune hemolytic anemia (AIHA) in children: Classification, clinical features, and diagnosis" and "Warm autoimmune hemolytic anemia (AIHA) in adults".)

●Paroxysmal nocturnal hemoglobinuria (PNH) – PNH is an acquired hematopoietic stem cell disorder that can cause intravascular hemolysis and anemia. Like unstable hemoglobin variants, individuals with PNH have unexplained DAT-negative hemolysis. Unlike unstable hemoglobin variants, individuals with PNH typically will have a record of a normal complete blood count in the past and may have more severe symptoms including pain from smooth muscle dystonia, thrombosis, and hemoglobinuria due to the large intravascular component of the hemolysis. (See "Clinical manifestations and diagnosis of paroxysmal nocturnal hemoglobinuria".)

MANAGEMENT — Unstable hemoglobins generally produce mild hemolytic anemia that does not require major interventions. Individuals with more severe symptoms may require transfusions and/or splenectomy. All individuals suspected of having an unstable hemoglobin should be referred to a hematologist, especially one practicing in a center with special expertise in red blood cell (RBC) disorders.

Hydroxyurea has stimulated fetal hemoglobin (Hb F) production in rare cases of unstable hemoglobins with severe hemolysis, but data regarding this practice are limited, the responses uneven, and its risks and benefits should be carefully weighed [33,49,50].

Reports have described hematopoietic cell transplantation in individuals with severe disease [51].

Folic acid — Folic acid is often prescribed for individuals with chronic hemolytic anemia to compensate for increased folate consumption in the setting of hemolysis. A typical dose is 1 mg per day orally. This may be unnecessary, as most high-income countries routinely supplement foods with folic acid, but costs and toxicities of additional folic acid supplementation are extremely low.

The possibility of folate deficiency may be evaluated in individuals not receiving folic acid who develop worsening of anemia, especially if associated with an increase in mean corpuscular volume (MCV) and a decrease in reticulocyte count.

Avoidance of oxidant drugs — Some individuals with an unstable hemoglobin develop increased hemolysis when exposed to oxidant drugs, and it is prudent to discontinue possible offending drugs in any individual who appears to have worsening hemolysis and/or when suitable alternatives are available. Implicated medications are generally similar to those that cause hemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. (See "Drug-induced hemolytic anemia", section on 'Oxidant injury'.)

Prevention and prompt treatment of infections — Some individuals with unstable hemoglobins have severe hemolysis in the setting of bacterial or viral infections. Routine preventive measures and age-appropriate vaccinations are used; these are discussed separately:

●Prevention – (See "Infection prevention: Precautions for preventing transmission of infection".)

●Vaccines for children – (See "Standard immunizations for children and adolescents: Overview", section on 'Routine schedule'.)

●Vaccines for adults – (See "Standard immunizations for nonpregnant adults".)

Prompt evaluation and treatment of infections is also appropriate.

Transfusions — Individuals with severe hemolysis and/or aplastic crisis (eg, in the setting of parvovirus B19 infection) may develop anemia severe enough to require RBC transfusion. These decisions are made on a case-by-case basis depending on the severity of symptoms, anemia, and bone marrow suppression.

Splenectomy — The question of splenectomy arises in individuals with more severe hemolysis. Although the spleen plays an important pathophysiologic role in the destruction of Heinz body-containing RBCs, the potential benefit of splenectomy must be weighed against the increased susceptibility to pneumococcal infections, risks of thrombosis and pulmonary hypertension, and the need for pneumococcal vaccines as well as prophylactic penicillin when splenectomy is performed during childhood. (See "Hereditary spherocytosis", section on 'Splenectomy'.)

On balance, splenectomy is often beneficial for the most severe cases, and partial correction of the anemia has been achieved in some instances [27]. However, for unknown reasons, splenectomy does not always reduce hemolysis or anemia.

Thromboembolic events have also been noted following splenectomy for unstable hemoglobin [52]. The etiology of the increased tendency to thrombosis in congenital hemolytic anemia is likely to be multifactorial, but postsplenectomy erythrocytosis, RBC procoagulant-containing microparticles, and post-splenectomy thrombocytosis may play some role.

Testing of family members and genetic counseling — Family members of an individual with an unstable hemoglobin documented by genetic analysis may be tested by standard hematologic evaluation such as complete blood count (CBC), reticulocyte count, and review of the peripheral blood smear, with high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE) if the picture is consistent with an unstable hemoglobin and that hemoglobin is separable by HPLC or CZE.

As noted above, the majority of unstable hemoglobins are transmitted in an autosomal dominant pattern. (See 'Overview of genetics' above.)

Additional information about genetic counseling is presented separately. (See "Genetic counseling: Family history interpretation and risk assessment".)

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: Sickle cell disease and thalassemias".)

SUMMARY AND RECOMMENDATIONS

●Definition and prevalence – Unstable hemoglobin variants are inherited mutations affecting globin genes; most are autosomal dominant and private (they affect only a single family/pedigree). Hemoglobin Köln and hemoglobin Zürich have been reported in several kindreds, and hemoglobin Hasharon is seen in individuals with Ashkenazi Jewish ancestry. (See 'Epidemiology' above.)

●Genetics and pathophysiology – Unstable hemoglobin variants can affect alpha, beta, gamma, or delta globin chains. They can disrupt folding, assembly, or subunit interactions in the globin molecules or interactions between heme and globin. Denatured hemoglobin precipitates in red blood cells (RBCs), which in turn may cause Heinz body formation and extravascular hemolysis as the spleen clears abnormal RBCs. (See 'Pathophysiology of hemolysis' above and 'Genetic variants and their effect on protein structure' above.)

●Presentation – Typical clinical manifestations include varying degrees of hemolysis, anemia, and manifestations of hemolysis (gallstones, splenomegaly, pigmenturia). Clinical variability depends on which globin chain is affected, whether the variant alters oxygen affinity, environmental factors (smoking, oxidant medications), and unidentified modifiers. (See 'Clinical manifestations' above.)

●Diagnosis – An unstable hemoglobin is suspected in a child or adult with unexplained hemolytic anemia. Presentations are usually in childhood or adolescence. However, many adults with chronic mild hemolytic anemia remain undiagnosed. Initial testing includes laboratory studies for hemolysis (table 1), review of the peripheral blood smear, and Heinz body preparation (if the laboratory has appropriate expertise). Specialized testing is pursued if the initial evaluation does not reveal another cause. Genetic testing is the most definitive means of establishing the diagnosis. (See 'Diagnostic evaluation' above.)

●Differential diagnosis – Several other causes of inherited and acquired hemolytic anemia, especially those associated with Heinz bodies, are described above. (See 'Differential diagnosis' above.)

●Management – Management is largely supportive and includes avoidance of oxidant drugs when possible, prevention and prompt treatment of infections, and possibly folic acid supplementation. Less commonly, transfusions and/or splenectomy may be needed. Testing of first-degree relatives is also appropriate. (See 'Management' above.)

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