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Hemoglobin and hemoglobinopathies
Abstract
Background
Disorders of hemoglobin (Hb) are collectively the commonest Mendelian disease found in humans. These disorders encompass both thalassemia and hemoglobinopathies. Thalassemia refers to quantitative deficiencies of one or more globin sub-units of the Hb molecule, with α-thalassemia and β-thalassemia being defined as reduced or absent production of α-globin and β-globin chains, respectively. Hemoglobinopathies are a qualitative defect resulting from globin gene mutations that change the amino acid sequence and lead to the production of Hb variants. Overlaps between the two forms of globin disorders occur. Some forms of β-thalassemia result from structural hemoglobin variants that are not effectively synthesized or are unstable, leading to a functional deficiency of β-globin chains and a thalassemic phenotype. Globin disorders are the first genetic diseases to be characterized at the molecular level and early work in the field opened up the era of molecular medicine. Thalassemia and hemoglobinopathies occur at the highest frequency in countries of the tropics, although they are seen at increasing frequencies among the emigrants from this region to other countries, hence causing significant health care burden worldwide.
Content
This chapter describes the protein structure, biosynthesis, switching, and physiologic function of hemoglobin. Emphasis is placed on the analytical methods, ranging from hematologic investigation and hemoglobin study to molecular methods of detection. Challenges and caveats on the interpretation of laboratory data are discussed. Clinical aspects of thalassemic syndromes and hemoglobinopathies are described to illustrate the significance of the laboratory studies.
Biochemistry, genetics, and physiology of hemoglobin
Hemoglobin is a hemoprotein whose primary function is to transport oxygen from the lungs to body tissues. It was first isolated in 1849, and was the first oligomeric protein to be characterized by ultracentrifugation and to have (1) its molecular mass accurately determined, (2) its physiologic function described, and (3) after the 25-year study of Perutz and colleagues in Cambridge, its structure defined by x-ray crystallography. In 1949, Pauling and colleagues showed that Hb from patients with sickle cell disease differed from normal Hb because it has two to four additional net positive charges. Later, the reasons for the charge difference were elucidated by locating the single amino acid difference between Hb from normal individuals and Hb from those with sickle cell disease.
Biochemistry
Hemoglobin is a globular protein with a diameter of 6.4 nm and a molecular mass of approximately 64,500 Da. As shown in Fig. 77.1 , Hb consists of four globin subunits (two α- and two non–α- [β-, γ-, or δ]-chains), with each looped around itself to form a pocket or cleft in which the heme group nestles. Normally, this heme pocket is formed entirely by nonpolar (hydrophobic) amino acids. The heme moiety (see Fig. 77.1 ) is suspended within this pocket by an attachment of its Fe atom to the imidazole group of the proximal histidine (position 92 of the β-chain [β92] or position 87 of the α-chain [α87]). The imidazole group of the distal histidine (β63 or α58) is also in contiguity with the Fe of heme, but it appears to swing into and out of this position to permit the passage of oxygen into and out of the Hb molecule. The four Fe atoms are in the divalent state, whether Hb is oxygenated or deoxygenated.
Protein structure
As with all proteins, the function of Hb is dictated by its primary, secondary, tertiary, and quaternary structures.
Primary structure.
The α- and non–α-globin chains of Hb are 141 and 146 amino acid residues in length, respectively. Some sequence homology has been noted, with 64 individual amino acid residues in identical positions in both α- and β-chains. The β-chain differs from the δ- and γ-chains by 39 and 10 residues, respectively. The amino terminal of the β-globin chain is the site of attachment of glucose (HbA 1c ), urea, and salicylate. The carboxy terminal amino acid of the β-chain is tyrosine and can function as a part of salt bridges. Although no disulfide bonds are present, six SH groups have been noted (from cysteine at positions α104, β93, and β112). The γ-chain has a glycine amino terminal, and the alkali resistance of Hb F is attributed to the presence of threonine and tryptophan at positions 112 and 130 of the γ-chain, respectively. The γ-chain is unique in that it is the only globin chain to be highly susceptible to acetylation, and acetylated Hb F is a prominent feature in cord and neonatal blood, and may form as much as 25% of the total Hb. The N-terminal valine of the β-globin chain of HbA 1c can also be acetylated to a smaller degree (<1 to 3%), for example, in alcoholic liver disease.
Secondary structure.
Approximately 75 to 80% of polypeptide chains of the α- and non–α-chains are arranged in helices, with the remainder forming nonhelical turns. The β-chain of Hb A is arranged into eight helices identified as A through H. In contrast, the α-chain is missing an equivalent of the D helix and has only seven helices. Nomenclature within the helices identifies the helix and the position within the helix of the amino acid residue (e.g., F3 is the third amino acid residue in the F helix). Amino acid residues in the peptide chains that join adjacent helices are described by the identification of two adjacent helices and the position of residues within the joining peptide. For example, EF3 would be the third residue in the peptide joining the E and F helices.
Tertiary structure.
The tertiary structure of Hb refers to the arrangement of helices into a three-dimensional, pretzel-like structure. The heme group, located in a crevice between the E and F helices, is attached to histidine residues in each globin chain. Heme iron is axially hinged to globin proteins by an invariant histidine residue in helix F8, termed the proximal histidine. The opposite axial position binds O 2 , which is stabilized by interaction with the conserved distal histidine in helix E7. This attachment is essential to maintaining the secondary and tertiary structure of the globin chains.
Quaternary structure.
The quaternary structure of Hb results from the attachment of the four globin chains to each other. Within the Hb tetramer, each globin subunit binds the unlike chain through two distinct interfaces, termed the α1β1 and α1β2. The globin dimers are formed through the extremely high affinity α1β1 interaction. Globin monomers are relatively unstable compared with dimers, which tend to precipitate inside the red cell and cause erythrotoxicity. Thus mutations that impair α1β1 interaction may cause hemolytic anemia. On the other hand, the α1β2 interaction is of lower affinity and mediates tetramerization. Oxygen binding destabilizes the α1β2 interaction that results in the transition of quaternary structure from the T state (tense, deoxygenated) to the R state (relaxed, oxygenated), which facilitates O 2 binding to additional subunits. This process causes cooperative O 2 binding as illustrated by the characteristic sigmoidal shape of the oxygen dissociation curve. Mutations within the α1β2 interaction region alter the functional property of Hb by disturbing O 2 binding characteristics. Cooperativity represents a general phenomenon termed allosteric regulation, in which effector molecules exert effect by binding to regions that are distinct from the active site. In addition to O 2 , another allosteric regulator H + binds Hb to promote O 2 release in a process termed the Bohr effect. The compound 2,3-biphosphoglycerate (2,3-BPG), formed as a result of glycolysis and present at relatively high concentrations in red blood cells, is another important allosteric regulator.
Modified hemoglobins
In addition to the Hbs discussed previously, carboxyhemoglobin, methemoglobin, and sulfhemoglobin are other Hbs whose structures have been environmentally or chemically modified.
Each of the modified Hbs has a characteristic spectral pattern, as shown in Fig. 77.2 . These spectral characteristics form the basis of analysis in the many co-oximeters and blood gas analyzers (see Chapter 37 ) that provide, in a single analysis, the simultaneous quantitative measurement of carboxyhemoglobin, methemoglobin, and sulfhemoglobin. The spectral scans are performed using multidiode arrays covering a number of wavelengths, followed by patented calculations that discriminate between normal and modified Hbs.
Carboxyhemoglobin
Carboxyhemoglobin is formed by the preferential attachment of carbon monoxide instead of oxygen to Hb. Carboxyhemoglobin concentrations (usually expressed as a carboxyhemoglobin saturation) have been known to reach 20% in individuals who are exposed to significant workplace concentrations of carbon monoxide. For example, police directing traffic at busy intersections and workers in radiator and welding shops have high carboxyhemoglobin concentrations at the end of the working day. The ability to perform heavy manual work or complex tasks is impaired at carboxyhemoglobin concentrations of 10% or even less for some individuals. Faulty home furnaces and automobile exhaust systems have been known to produce large amounts of carbon monoxide, sometimes with tragic results. Carboxyhemoglobin saturation that varies from 15 to 25% may be associated with dizziness, headaches, and nausea, and greater than 50% saturation is considered life threatening. After removal of the exposed individual from the carbon monoxide source, a slow decline in carboxyhemoglobin saturation occurs, in keeping with the half-life of 4 to 5 hours at sea level.
Methemoglobin
The Fe of heme is normally in the reduced ferrous state (Fe 2+ ). Under alkaline conditions, the Fe is oxidized to the ferric state (Fe 3+ ) by toxic agents, such as nitrates (found in some well waters), aniline dyes, chlorates, drugs (e.g., quinones, phenacetin, and sulfonamides), or local anesthetics (e.g., procaine, benzocaine, and lidocaine). This oxidation converts the heme to hematin and the Hb to methemoglobin. Patients with methemoglobin are cyanotic because ferric hemes of methemoglobin bind oxygen irreversibly. These patients are labeled as pseudocyanotic since the O 2 saturation of Hb is low despite adequate arterial oxygenation. Low levels of methemoglobin are normally reduced to Hb in the cell by the reduced form of the nicotinamide-adenine dinucleotide–cytochrome reductase system.
Hereditary methemoglobinemia is a rare condition that was first described in Europeans, but was later found in individuals of many racial backgrounds. Familial methemoglobinemia in an autosomal recessive mode of transmission is due to a deficiency in the enzyme nicotinamide-adenine dinucleotide–cytochrome b5 reductase. Hb variants, Hb M Saskatoon, Hb Freiburg, and Hb St. Louis stabilize the ferric Fe state and are associated with an autosomal dominant familial methemoglobinemia. The presence of methemoglobin in unexplained cyanosis can be identified through measurement of its absorption spectrum, either by simple spectroscopy or more definitely by spectrophotometry. Methemoglobinemia is treated by the administration of methylene blue. Ascorbic acid is less effective and may be used where methylene blue cannot be used.
Sulfhemoglobin
Sulfhemoglobin is produced by the reaction of sulfur-containing compounds with heme to form an irreversible chemical alteration and oxidation of Hb by the introduction of sulfur in one or more of the porphyrin rings. The most common cause of sulfhemoglobinemia is exposure to drugs, such as phenacetin and sulfonamides. Sulfhemoglobin cannot transport oxygen, and cyanosis is noted at low concentrations.
Biosynthesis
The biosynthesis of Hb requires the biosynthesis of both heme and the globin polypeptide chains.
Heme biosynthesis
Heme, ferrous protoporphyrin IX, consists of four pyrrole rings surrounding an iron (Fe) atom with four of the six electron pairs of Fe attached to the nitrogen atoms in the pyrrole rings (see Chapter 40 ). One of the remaining electron pairs attaches to a histidine residue in a globin chain, and the other pair is available for binding and transporting an oxygen molecule. The latter electron pair is protected from oxidation by the surrounding nonpolar amino acid residues of the globin chain. Hemin results from the relatively easy oxidation of the Fe of heme, from the ferrous to the ferric state. To remain electrically neutral, a halide molecule, usually chloride, becomes attached to hemin. In alkaline solution, hematin is formed by the replacement of the halide atom of hemin by a hydroxyl group.
The biosynthesis of heme, shown schematically in Fig. 77.3 , takes place primarily in the bone marrow and the liver, and is an eight-step process, with each step involving a different genetically controlled enzyme. Details of this process are given in Chapter 40 .
Heme synthesis is controlled by a regulatory negative feedback loop that relies on post-translational regulation through the interaction of iron-regulatory proteins (IRP) with iron-responsive elements (IRE) on the messenger ribonucleic acid (mRNA) that encodes iron transporters, ferroproteins, and enzymes involved in cellular iron homeostasis. Excess heme inhibits the activity of ferrochelatase and the acquisition of Fe from the transport protein transferrin. The decrease in Fe acquisition leads to a decrease in Fe uptake into the cell, with a subsequent decrease in δ-aminolevulinic acid and heme production. Conversely, Fe deficiency and increased erythropoietin synthesis lead to the combination of the IRP with the IRE in the 3′-end of the transferrin receptor protein mRNA. This combination in turn leads to protection of the mRNA from degradation with subsequent increased uptake of Fe into erythroid cells caused by the increased expression of transferrin receptors on the cell membrane.
Globin synthesis and globin gene families
The genes that control the α-like and ζ-globin chains are located in a cluster on chromosome 16 at position 16p13.3, which is near the chromosome 16 telomere ( Fig. 77.4 ). The α-like gene extends more than 28 kb, and contains, reading from the upstream (5′) end to the downstream (3′) end of the DNA segment, an embryonic α-like ζ-globin gene, a hypervariable region, a pseudo(ψ)–ζ-gene, a pair of pseudo (ψ)–α-genes, a pair of functional α-globin genes, an unexpressed α-like θ gene, and finally, another hypervariable region. Approximately 25 to 65 kb upstream of the α-globin genes are four highly conserved noncoding sequences known as multispecies conserved sequences (MCS) and termed MCS-R1 to MCS-R4, which are involved in the regulation of the α-globin genes. Of these elements to date, only MCS-R2, also known as HS-40, has been shown to be essential for α-globin gene expression. Alpha-thalassemia arises from the deletion of one or more α-globin genes or point mutations. Deletion of all four genes results in the condition of Hb Bart hydrops fetalis and is typically incompatible with life. Substantial genetic variability is seen between individuals and ethnic groups with respect to the copy number of the ζ-, ψζ-, and α-genes.
The β-, γ-, and δ-globin genes are clustered closely together on chromosome 11. Reading from the 5′ end, the gene sequence is an e-gene followed by two γ-genes (designated Gγ and Aγ, respectively), a pseudo–ψβ-gene, a δ-gene, and a β-gene. Therefore two genes encode the γ-chain, with one gene each encoding the δ- and β-chains. Upstream to the β-globin locus is the locus control region (LCR), consisting of several DNase I hypersensitive sites that contain binding motifs for transcription activators. The LCR functions as an enhancer to regulate the spatiotemporal transcription of the globin genes. When the globin gene is actively transcribed, the LCR hypersensitive sites are positioned in close proximity to the active gene, forming a chromatin loop.
The globin genes have three exons and two introns with a promoter region (specific for the globin chain) at the 5′ end of each gene. This structure has been highly conserved throughout evolution. The upstream regions flanking the first exon contain a number of sequence motifs that are necessary for specifying correct transcriptional initiation, such as a TATA box found 30 bp upstream of the initiation site, and one or more CCAAT sites at 70 bp upstream. The gene promoters also contain a CACCC or CCGCCC box that binds erythroid Krüppel-like factor 1, and some have binding sites for erythroid transcription factor GATA-1. In model systems, mutations introduced into such sequences lead to reduction in the level of transcription and are known as transcriptional mutants.
Hemoglobin switching
In normal human adults, Hb A is composed of two normal α- and two normal β-polypeptide chains and is represented symbolically as α 2 β 2 ( Table 77.1 ); it represents at least 96% of the total Hb. Hb A 2 is typically approximately 2.5 to 3.5% of total Hb; it contains two α- and two δ-chains, and is designated as α 2 δ 2 . HbA2′ is considered a variant of Hb A 2 and is the result of a glycine-to-arginine substitution in the 16th position of the δ-chain; it occurs in 1 to 2% of African Americans. It rarely forms more than 3% of the total Hb and has no clinical implication. Fetal Hb (Hb F) predominates during fetal life but rapidly diminishes during the first year of postnatal life. In normal adults, less than 1% of Hb is Hb F. It consists of two α- and two γ-chains (α 2 γ 2 ).
TABLE 77.1
Hemoglobins in Embryonic, Fetal, and Adult Life
| GLOBIN CHAINS | |||
| Stage of Development | α-Cluster | β-Cluster | Hemoglobins |
| Embryonic | ζ, α | ε |
|
| Fetal | α | γ | Hb F (α 2 γ 2 ) |
| Adult | α | β, γ, δ |
|
In early embryonic life, the yolk sac produces the globin chains ζ and e (see Table 77.1 ). These globin chains combine to form the major embryonic Hbs, Hb Gower 1 (ζ 2 ε 2 ) and 2 (α 2 ε 2 ), and Hb Portland 1 (ζ 2 γ 2 ) and 2 (ζ 2 β 2 ). Production of the ζ-chain ceases at the gestational age of approximately 4 months.
Production of α- and γ-chains starts at approximately 6 weeks’ gestation, with Hb F (α 2 γ 2 ) increasing in concentration to become the major Hb found in the fetus ( Fig. 77.5 ). Glycine or alanine may be found at position 136 of the γ-chain in the fetus, giving rise to two distinct γ-chains designated Gγ and Aγ, respectively. Formation of Hb A (α 2 β 2 ) commences at approximately 28 weeks of gestation, and at birth it can form up to 15% of the total Hb, with the remainder of the Hb consisting mainly of Hb F with a small amount of Hb A 2 . Production of the γ-chain declines after birth, and normal adult Hb F concentrations are usually obtained by 1 year of age but may be elevated until 2 years of age.
Physiologic role
The Fe of heme is in the ferrous state and is able to combine reversibly with oxygen to act as the major oxygen-carrying moiety. The term cooperativity is used to describe the interaction of globin chains in such a way that oxygenation of one heme group enhances the probability of oxygenation of the other heme group. The Bohr effect 8 refers to reduction of oxygen affinity due to a decrease in pH from the physiologic range (7.35 to 7.45) to 6.0, and is another result of this cooperativity. Because the pH of the tissue decreases as a result of the presence of the end products of anaerobic metabolism, carbon dioxide (CO 2 ) and carbonic acid, the delivery of oxygen to the exercising tissue is enhanced. The oxygen dissociation curve of normal blood Hb is sigmoidal ( Fig. 77.6 ). Physiologically, the CO 2 reversibly combines with the amino terminal groups of Hb to form carbamated Hb, which facilitates the removal of approximately 10% of the CO 2 that forms because of metabolism in the tissue to the lungs. Removal and transport of CO 2 from the tissue are enhanced by the preference for the attachment of more CO 2 by carbamated Hb.
Clinical significance of hemoglobin disorders
The thalassemia syndromes and hemoglobinopathies are clinical disorders related to Hb. They are collectively the commonest Mendelian or single gene disorders in the world. Although their clinical manifestations may overlap, they form two distinct groups of genetic disorders. Thalassemia refers to quantitative deficiency of one or more globin subunits of the Hb molecule. The name thalassemia is derived from the Greek word for “sea,” thalassa, because early cases of β-thalassemia were described in children of Mediterranean origin. Hemoglobinopathy is a qualitative defect resulting from globin gene mutations that change the amino acid sequence and lead to the production of Hb variants.
Thalassemia syndromes
Thalassemias are identified by the globin chain in which a production deficiency occurs. For example, α- and β-thalassemias result from a deficiency in α- or β-globin chain production, respectively ( Box 77.1 ). They are further clinically classified depending on the extent of globin chain production deficit and the resultant severity of the anemia. All the thalassemias have a similar pattern of inheritance: in most cases the gene defects are transmitted in a Mendelian autosomal recessive fashion. Thus the severe symptomatic clinical forms result from the inheritance of homozygous or compound heterozygous genotypes. The inheritance of α-thalassemia is more complicated because it involves the products of the linked pairs of α-globin genes (αα).
α-Thalassemias
The α-Thalassemias arise from deficiencies in production of the α-globin chains and are caused by deletions or (less frequently) point mutations in one or more of the four α-globin genes. The conventional nomenclature for the point mutations of an α-gene is “α T α,” and for deletion, it is “−α.” Deletion of one α-globin gene (−α/) is termed α + -thalassemia (or α-thal-2) while deletion of two α-globin genes on the same chromosome (− −/) is termed α 0 -thalassemia (or α-thal-1). Point mutations are much less frequent. The clinical spectrum of α-thalassemias correlates well with the number of the affected α-genes (i.e., from normal to the loss of all four genes). The inheritance of one or two α-globin gene deletion or mutation in various genotypic configuration results in a “α-thalassemia minor” (−α/αα; − −/αα; −α T /αα; α T α/−α; −α/−α). In general, carriers of such genotypes are asymptomatic and may range from hematologically silent to hypochromic microcytic red cell indices in association with a normal Hb level or slight anemia. The peripheral blood smear is quite variable, showing various degrees of hypochromia with some target cells and occasional poikilocytes. In carriers of α 0 thalassemia (− −/αα), it is possible to observe a few red cells that contain Hb H inclusions after supravital staining, which are β 4 tetramers that are due to an excess of the β chains. The Hb concentration in adult carriers of α + or α°-thalassemia can be indistinguishable from normal, but the percentage of Hb A 2 is slightly lower. Since α-chains bind more readily to β-chains than to the positively charged δ-chains, the HbA 2 level is decreased in limiting levels of α-chains as seen in α-thalassemia. Traces of Hb Bart’s (γ 4 ) in the neonatal period are detectable in a large proportion of neonates with α-thalassemia, and they decline during the first 6 months after birth. The Hb Bart’s level in cord blood allows the detection of α + -thalassemia carrier (<2% Hb Bart’s), α 0 -thalassemia carrier (2–5% Hb Bart’s) and Hb H disease (20 to 40% Hb Bart’s). In the California Newborn Screening Program, screening for Hb H disease is based on an elevated level (>25%) of a fast-eluting peak on high-performance liquid chromatography (HPLC), which is then subject to confirmation by DNA study. This program shows that α-thalassemia syndromes such as Hb H disease have become the most common nonsickle hemoglobin disorder in California.
Alpha-thalassemias are common in areas where β-thalassemia is also found at a high frequency. Thus the coinheritance of α- and β-thalassemia traits may occur and even ameliorate the hematologic parameters. Depending on the thalassemia status of the partner, subjects who are concurrent α- and β-thalassemia carriers are potentially at risk of being parents to offspring affected by β-thalassemia major, Hb H disease, and Hb Bart’s hydrops fetalis. In some cases for genetic counseling in families in whom α- and β-thalassemias are present, genotype diagnosis is essential to fully prevent hydrops fetalis since the α 0 -thalassemia is masked in carriers of concurrent α- and β-thalassemias. ,
Point mutations of the α-globin gene, although less common than deletions, are important since their coinheritance with α°-thalassemia may result in nondeletional Hb H disease. An example is the chain termination mutation of the α-globin gene, Hb Constant Spring (α142 Gln) or Hb CS . The unstable mutant Hb CS causes not only a reduction in α 2 globin expression from the affected chromosome, but also red cell membrane damage by the partially oxidized α CS chains in addition to the excess β chains; therefore the carriers, but particularly the homozygotes, have a more severe phenotype than α-thalassemia minor, but it is not as severe as most cases of Hb H disease ( Table 77.2 ).
TABLE 77.2
The Alpha-Thalassemias
Data from Vichinsky EP. Alpha thalassemia major–new mutations, intrauterine management, and outcomes. Hematology Am Soc Hematol Educ Program. 2009:35–41.
| Condition | Affected Number of Alpha-Globin Genes | Phenotype |
|---|---|---|
| Silent carrier | One gene affected | Asymptomatic or occasional low red blood cell indexes |
| Alpha-thalassemia trait | Two genes affected | Mild microcytic and hypochromic anemia |
| Hb H disease | Three genes affected | Mild-to-moderate anemia, splenomegaly, jaundice |
| Alpha-thalassemia major/Hb Bart’s hydrops | Four genes affected | Most severe form Hb Bart’s hydrops Death in utero or at birth |
Hb , Hemoglobin.
The α-thalassemias occur worldwide and are particularly prevalent in Southeast Asia, Southern China, Mediterranean countries (particularly Greece and the Greek Cypriot part of Cyprus), India, the Middle East, and the islands of the South Pacific. Individual types of α-thalassemias are discussed in the following sections and reviewed in the literature.
Hemoglobin bart’s hydrops fetalis (α-thalassemia major)
Hemoglobin Bart’s hydrops fetalis results from deletion of all four α-globin genes. There is the subsequent inability to produce any α-globin chains, leading to failure of synthesis of Hb A, F, or A 2 . In the fetus, an excess number of γ-globin chains join together to form unstable tetramers known as Hb Bart’s (γ 4 ), named after the London hospital where it was discovered. Mothers who carry a fetus with Hb Bart’s hydrops fetalis usually present clinically between 20 and 26 weeks of gestation with pregnancy-induced hypertension and polyhydramnios. Ultrasound of the fetus shows hydrops. Fetal anemia is suggested by an increase in the peak systolic velocity in the middle cerebral artery compared to gestation-specific reference interval on Doppler measurement. Severe fetal anemia (Hb usually <80 g/L) is noted on a fetal blood sample obtained by cordocentesis. It is important to rule out other causes for the hydropic fetus by performing serologic testing for infection such as toxoplasmosis, rubella, cytomegalovirus, herpes simplex, Parvovirus B19, and for red cell alloantibodies if hemolytic disease of the fetus is suspected.
HPLC analysis of a cordocentesis blood sample shows one or two very sharp and narrow peaks at the injection point on the chromatogram ( Fig. 77.7 A). The major band is Hb Bart’s with a smaller band attributed to Hb Portland. Complete absence of Hb F is noted. Alkaline electrophoresis shows a band migrating at the anodal position (Hb Bart’s), with another band in the Hb A position (Hb Portland) ( Fig. 77.8 )
Hb Bart’s hydrops fetalis is almost invariably fatal if left untreated, with some fetuses dying in utero, and others surviving a few hours after birth. Treatment using intrauterine transfusion may be able to salvage the fetus, but risks of complications including growth retardation and severe brain damage remain, which may be related to hypoxemia following long-standing intrauterine anemia.
Laboratory investigation of the parents of fetuses with Hb Bart’s hydrops fetalis shows a normal HPLC pattern with normal Hb F and A 2 quantification. Parental analysis typically shows a decreased concentration of Hb and decreased MCH and MCV, with the blood smear showing hypochromic, microcytic red cells. The Hb H inclusion body test is usually positive in the parents. A two α-gene cis -deletion (−−/αα) or a three gene deletion (−−/−α) is seen in genetic testing of both parents. This requirement restricts the incidence of Hb Bart’s hydrops fetalis to a much smaller population than would be expected based on the worldwide distribution of α-thalassemias because the presence of trans- deletions (−α/−α) in both parents would not give rise to a four gene deletion in the offspring. Hb Bart’s hydrops fetalis is relatively common in Southeast Asia, particularly in Thailand, the Philippines, and Hong Kong, where there is a high prevalence of the -- SEA /(SEA) deletion. Deletions that remove the embryonic ζ-globin gene together with both α-globin genes (e.g. – FIL , – THAI ) can lead to early death of the embryo and usually presents as missed abortions.
Hemoglobin H disease (α-thalassemia intermedia)
This disorder is usually caused by a three α-globin gene deletion (− −/−α) and is characterized by a chronic anemia of variable severity. Individuals with nondeletional Hb H disease (α T α/− −) are usually more severely affected and are more likely to require transfusion therapy than those with deletional Hb H disease. Significant underproduction of α-globin chains occurs with subsequent joining of free β-globin chains to form the insoluble β-globin chain tetramer Hb H. HPLC analysis of a hemolysate from an individual with Hb H disease shows two bands with short retention times forming a doublet together with a normal Hb A band. Hb F concentration is within the reference interval (see Fig. 77.7 D) but the Hb A 2 level is reduced. Electrophoresis at alkaline pH shows a fast-moving band together with a band in the Hb A position that possibly has reduced staining compared with other samples run concurrently. The complete blood count (CBC) shows a moderately reduced concentration of Hb, markedly reduced MCV and MCH, and a markedly increased red cell distribution width (RDW). RBC count is normal or slightly raised. Fe studies are normal, although the ferritin concentration may be elevated. However, the use of automated cell counters may flag adult patients with Hb H disease as iron deficiency, and vigilance should be exercised to avoid misdiagnosis. A blood film after staining with brilliant cresyl blue shows many red cells with inclusion bodies ( Fig. 77.9 A). Hb H disease, according to a recent clinical classification, can be considered a non–transfusion-dependent thalassemia. Fe therapy is not indicated, and transfusion therapy is usually unnecessary except for during an acute illness or in pregnancy. Red cell aplastic crisis is an uncommon complication during an acute Parvovirus B19 infection. Genetic counseling is recommended to prospective parents at risk of having an offspring with Hb H disease.
α-Thalassemia minor
α-Thalassemia minor is the result of two α-chain gene deletions. These deletions may be seen on the same chromosome (− −/αα, heterozygous α°-thalassemia or α-thal-1), described as a cis deletion, or on different chromosomes (−α/−α, homozygous α + -thalassemia or α-thal-2), described as a trans deletion. The CBC of affected individuals shows a mildly reduced Hb with low MCV and MCH. HPLC analysis shows no abnormal Hb peak, and Hb F and Hb A 2 concentrations are within the reference intervals. Fe studies are normal. The blood film shows occasional red cells with Hb H inclusions on supravital staining in most subjects with heterozygous α°-thalassemia, but they are rarely seen in heterozygous or even homozygous α + -thalassemia. Therefore this test is not performed in many parts of the world where α°-thalassemia is not prevalent and the diagnosis of α-thalassemia minor is based on exclusion criteria rather than definitive tests in many routine clinical laboratories. After exclusion of Fe deficiency, the presence of hypochromic microcytic red cell indexes in a patient with normal Hb A 2 and Hb F quantification forms the basis for a presumptive diagnosis of α-thalassemia minor, particularly in the setting of a positive family history. Definitive diagnosis of an α-thalassemia trait (α + or α°-thalassemia) can be achieved by DNA analysis using different methods (gap-polymerase chain reaction [PCR], multiplex ligation-dependent probe amplification [MLPA], allele specific oligonucleotide hybridization [ASO] sequencing). Molecular diagnosis is required for prenatal diagnosis in couples at risk for Hb Bart’s hydrops fetalis.
Silent α-thalassemia trait
A silent α-thalassemia trait results from a single α-globin gene deletion or mutation (−α/αα; α T α/αα). A single α-globin gene deletion or mutation is frequently clinically and hematologically silent. A CBC of an individual with this trait shows a normal or marginally decreased Hb concentration, MCV, and MCH. Fe studies are normal, and no abnormal Hb peaks are seen on HPLC analysis.
The effects of various α-thalassemias on the percentage of β-chain Hb variants are shown in Table 77.3 . In general, the percentage of the Hb variant decreases as a percentage of total Hb as the number of α-globin gene deletions increases. The proportion of β-chain variants is affected by charge. Positively charged β-chain Hb variants such as Hb S, C, D-Los Angeles, and E constitute less than half of the total Hb in the heterozygous state and are reduced further in the presence of α-thalassemia, due to increased competition between normal and mutant β-chain for the limited amounts of α-chain. Conversely, negatively charged β-chain Hb variants such as Hb J-Baltimore and J-Iran, in conjunction with α-thalassemia, show an increased proportion of the mutant Hb since the β-chain variant out-competes normal β-chain for the limited amounts of α-chain in the presence of α-thalassemia.
TABLE 77.3
Effects of α-Thalassemia on the Percentage of β-Chain Variant Hemoglobin in Heterozygotes
Modified from Bunn HF, Forget BG, eds. Hemoglobin: molecular genetic and clinical aspects. Philadelphia: Saunders; 1986.
| AS | AC | AE | |
|---|---|---|---|
| αα/αα | 41.0 ± 1.8 | 43.8 ± 1.5 | 30.0 ± 1.5 |
| αα/α− | 35.4 ± 1.6 | 37.5 ± 1.4 | 27.0 ± 2.0 |
| α − /α − or αα/ − | 28.1 ± 1.4 | 32.2 ± 0.8 | 22.0 ± 2.0 |
AS , Carrier of hemoglobin (Hb) S; AC , carrier of Hb C; AE , carrier of HbE.
β-Thalassemias
The β-thalassemias result from a reduction in the synthesis of the β-globin chain and are commonly found in (1) the Mediterranean region, (2) Africa, (3) the Middle East, and (4) Southeast Asia, especially the southern provinces of China, including Hong Kong, the Indian subcontinent, the Malay peninsula, Myanmar (Burma), and Indonesia. The frequency of gene distribution is estimated at 3 to 10% in some populations. The high frequency of β-thalassemia in the tropics is believed to reflect a survival advantage of heterozygotes against Plasmodium falciparum malaria. With increasing migration, β-thalassemia, once considered a rare genetic disease in Northern Europe, Australia, and North America, is now becoming more common all over the world. More than 250 β-thalassemia mutations have been described; however, in each ethnic group, a relatively small number of mutations account for most cases (the ratio most often quoted is that ≤20 mutations account for ≥80% of cases). The β-thalassemia alleles can be classified into β 0 -thalassemia, in which there is no β-globin gene production, and β + or β ++ -thalassemia, in which there is marked or mild reduction in the production of β-chains, respectively. Most are point mutations, small insertion or deletions within the gene or its immediate flanking sequence. They may affect any level of gene expression and are categorized according to the mechanism by which they affect gene function, namely transcription, RNA processing, and RNA translation.
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a. Mutations that affect transcription can either involve the β-globin gene promoter or the stretch of 50 nucleotides in the 5′-untranslated region (5′-UTR). Generally speaking, they result in a minimal to mild deficit in the output of β-globin chains as reflected by the relatively mild β + -thalassemia phenotype of those affected.
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b. Mutations that affect RNA processing can involve either of the invariant dinucleotides (GT at 5′ and AG at 3′) at the splice junction in which case normal splicing is abolished in entirety, resulting in the phenotype of β 0 -thalassemia. Mutations within the consensus sequences that flank the splice junctions reduce efficiency of normal splicing to varying degrees, producing a β + -thalassemia phenotype that ranges from mild to severe. Other splice mutations involve base substitutions within introns or exons. For instance, a cryptic splice site sequence of GT GGT GAG G is found in exon 1 spanning codons 24 to 27. Three mutations within this region activate this cryptic site, causing it to serve as an alternative donor site in RNA processing. The Hb E mutation (β26 GAG→AAG; Glu→Lys) also activates this cryptic splice site causing abnormal RNA processing so that normal splicing which produces Hb E variant is reduced. Other RNA processing mutants affect the polyadenylation signal (AATAAA) and the 3’-UTR. These are usually mild β + -thalassemia alleles.
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c. Mutations that affect mRNA translation at either the initiation or extension phases of globin synthesis are associated with a β 0 -thalassaemia phenotype. Roughly half of the β-thalassemia alleles are characterized by premature termination of β-chain extension. They result from introduction of termination codons due to frameshift or nonsense mutations and nearly all terminate within exons 1 and 2. Terminations early in the sequence are associated with minimal steady state levels of β-mRNA in erythroid cells, due to accelerated decay of the abnormal mRNA referred to as nonsense-mediated decay (NMD). In contrast, exon 3 mutations usually are not subject to NMD and hence result in substantial amounts of mutant mRNA with respect to normal mRNA, which presumably is translated into highly unstable variant β-chains and can lead to severe hemolysis and a dominantly inherited phenotype. Precipitation of unstable β-chains and concurrent excess α-globin chains also overload the intracellular proteolytic mechanisms of erythroblasts in the bone marrow, which aggravates ineffective erythropoiesis.
A few β-thalassemia mutations that segregate independently of the β-globin gene cluster have been described, presumably involving trans -acting regulatory factors. An updated list of these mutations is accessible at the Globin Gene Server Website ( http://globin.cse.psu.edu ). , Simple deletions of the β-globin gene are rare, ranging in size from 290 bp to more than 100 kb and are necessarily β 0 -thalassemias. The 619-bp deletion at the 3′ end of the β-globin gene is relatively common among Sindhi and Punjabi populations in India and Pakistan, whereas the 100-kb Chinese ( A γδβ)° deletion which includes the δ and β-globin gene are relatively common in Southern Chinese populations. Some deletions are associated with an unusually high level of Hb A 2 or Hb F. Large deletions that affect the entire β-globin gene cluster (eγγδβ)°are rare and restricted to single families.
The clinical classification of β-thalassemia includes thalassemia major (TM; transfusion-dependent), thalassemia intermedia (TI; of intermediate severity, nontransfusion-dependent), and thalassemia minor (asymptomatic). The severity of the clinical manifestations correlates well with the degree of imbalance of globin chains, depending on the β-globin gene defects and their interaction. The production of β-globin chains is quantitatively reduced to different degrees, whereas the synthesis of α-globin continues as normal, resulting in accumulation of excess unmatched α-globin chains in the erythroid precursors. Clinical manifestations of β-thalassemia range from mild anemia to severe life-threatening disease that requires lifelong transfusions ( Fig. 77.10 ).
β -thalassemia major
This is sometimes called Cooley’s anemia, after the physician who first described the condition in 1925 in the children of Italian and Greek immigrants in New York.
β-Thalassemia major (TM) results from homozygous or compound heterozygous β 0 -thalassemia mutations that severely interfere with RNA splicing or translation. Mutations that interfere with translation account for almost 50% of all β-thalassemia mutations.
Clinical presentation usually occurs at younger than 1 year of age, with features such as small size for age, abdominal girth expansion, and failure to thrive. Physical examination of the patient may reveal frontal bossing (an unusually prominent forehead) caused by thickening of the cranial bones, pallor, and prominence of the cheek bones, which in older children obscures the base of the nose and exposes the teeth. These features are a result of marrow expansion (up to a 30-fold increase) caused by ineffective erythropoiesis with production of highly unstable α-globin tetramers, leading to a sequence of events responsible for bone marrow expansion, anemia, hemolysis, splenomegaly, and increased Fe absorption.
Typical CBC results include severe anemia with Hb concentration between 30 and 65 g/L, MCV of 48 to 72 fL, and MCH of 23 to 32 pg. A characteristic markedly abnormal RBC morphology is noted on the peripheral blood smear; this includes a large number of microcytes and/or macrocytes, prominent basophilic stippling, numerous target cells, which may have a bridge joining the central and peripheral pigment zones, polychromasia, and occasional spherocytes, schistocytes, and nucleated red cells. Circulating nucleated red cells often poorly hemoglobinized are rather characteristic of β-thalassemia major or intermedia. Typical peripheral blood on a patient with β° thalassemia major is shown in Fig. 77.11 .
White blood cell (WBC) and platelet counts are usually normal. Ferritin is usually within the upper half of the reference interval at the time of diagnosis, and total bilirubin is mildly elevated mainly due to elevation in the unconjugated fraction. Urinalysis frequently shows increased urobilinogen or urobilin concentration, and urine is often dark brown to black because of the presence of dipyroles and mesobilifuscin. The latter features reflect ineffective hematopoiesis with intramedullary red cell destruction. HPLC analysis (see Fig. 77.7 B) shows a major Hb F peak with absence of an Hb A peak and variable Hb A 2 (reference interval, 1 to 5.9%; mean, 1.7%) peak. Electrophoresis at alkaline and acid pH shows a dominant band in the F position in both gels.
Family studies on both parents and siblings should be performed, and the classical β-thalassemia minor pattern, described later in the chapter, should be found in the parents. Siblings may be normal or may have β-thalassemia minor. A family case history is seen in Fig. 77.12 .
The conventional treatment for TM patients includes regular transfusion therapy and Fe chelation. Many patients with TM require splenectomy because of hypersplenism. However, optimal clinical management may delay or even obviate the need for splenectomy, which was common in the past. After splenectomy, inclusion bodies consisting of denatured α-chains (also termed Fessas bodies) can be observed in the blood smear after staining with methyl violet. Fe overload is an inevitable and serious complication of long-term blood transfusion therapy that requires adequate treatment to prevent early death mainly from Fe-induced cardiac disease. Puberty is often delayed, incomplete, or completely absent. In boys, active spermatogenesis may occur, and Leydig cell function is normal. The quality and duration of life of TM patients has been transformed over the last 20 years, with life expectancy increasing well into the fourth and fifth decades. Many children who are adequately transfused and are fully compliant with Fe chelation therapy develop normally, enter puberty, and become sexually mature. The availability of oral Fe chelators, such as Deferiprone (ApoPharma, Toronto, Canada) and Deferasirox (Novartis, Basel, Switzerland), has significantly improved the adherence to chelation, which has affected survival. Nevertheless, prolongation of life is accompanied by several complications, partly due to the underlying disorder and partly as a consequence of the treatment with blood transfusions and Fe overload, such as diabetes mellitus, hypothyroidism, hypoparathyroidism, and liver disease.
Allogeneic hemopoietic stem cell transplantation is currently the only method available to cure transfusion-dependent TM; it has been used worldwide. Gene therapy and other innovative therapeutic modalities for the cure of TM are under investigation.
β -thalassemia intermedia
β-Thalassemia intermedia (TI) is a clinical term used to describe patients with anemia and splenomegaly, but who do not have the full spectrum of clinical severity found in β-TM. The clinical phenotypes of β-TI lie between those of thalassemia minor and major, encompassing a wide clinical spectrum. Mildly affected patients are almost completely asymptomatic until adult life, experiencing only mild anemia and spontaneously maintaining Hb levels between 7 and 10 g/dL (70 and 100 g/L). Patients with more severe TI generally present between the ages of 2 and 6 years, and although they are able to survive without regular transfusion therapy, growth and development can be retarded. Most β-TI patients are homozygotes or compound heterozygotes for β-gene mutations (β + /β + ; β°/β + ). Because the clinical severity of the disease is dictated by the different extent of globin chain imbalance, at least three different mechanisms may promote the milder clinical characteristics of TI compared with TM: inheritance of mild or silent β-gene mutations; coinheritance of determinants associated with increased γ-chain production that contributes to neutralizing the large proportion of unbound α-chains; and coinheritance of α-thalassemia that reduces the synthesis of α-chains, thereby reducing the α/non–α-chain imbalance. Increased Hb F production not only improves globin chain imbalance, but also raises the total hemoglobin level. Other molecular mechanisms of β-TI include compound heterozygosity for β-thalassemia and δβ-thalassemia or hereditary persistence of fetal hemoglobin (HPFH), compound heterozygosity for β-thalassemia and Hb E, and simple heterozygosity for β-thalassemia with co-existing amplification of α-globin genes.
β-TI is clinically labeled as non–transfusion-dependent thalassemia (NTDT) because the affected patients do not require lifelong regular transfusions for survival, although they may require occasional or even frequent blood transfusions in particular situations such as pregnancy, surgery, and infections.
HPLC analysis shows a variable Hb F peak with a reduced Hb A peak. Hb A 2 is usually increased. Bands in the A and F positions are seen on electrophoresis at both alkaline and acid pH. Hb is significantly reduced to values between 6 and 10 g/dL (60 and 100 g/L). The peripheral blood smear shows the same features seen in β-TM, including anisocytosis, hypochromia, target cells, basophilic stippling, and nucleated RBCs.
β -thalassemia minor ( β -thalassemia trait)
Subjects with β-thalassemia minor are asymptomatic. The CBC shows low normal or mildly decreased Hb concentration and hematocrit (Hct), decreased MCV (<80 fL) and MCH (<25 pg), and normal to slightly increased RDW. The peripheral blood smear shows microcytic hypochromic RBCs with occasional basophilic stippling and target cells. Note that for patients with liver disease, megaloblastic anemia or myelodysplastic syndrome in association with concomitant underlying β-thalassemia, the MCV value may be spuriously normalized. If these combinations are suspected, careful examination of the blood smear for dimorphic red cell populations is warranted.
The diagnosis of β-thalassemia minor, with appropriate indexes in the CBC, is dependent on the finding of a raised Hb A 2 (≥4%). Even when the quality of measurement is assured, there are subjects in whom Hb A 2 level is borderline raised, typically in the range of 3.1 to 3.9%. Recently, it was found that a significant proportion of these individuals may have underlying heterozygous KLF1 mutations. However, there are rare β-thalassemia carriers who show normal to borderline raised Hb A 2 level, often due to coinheritance of linked β-globin gene and δ-globin gene mutations. Certain mild to silent β-thalassemia mutations, such as those involving the promoter or 5′ untranslated region, may be associated with normal to mildly reduced MCV and a borderline raised Hb A 2 level. The clinical relevance of these mutations is that, if the partner is having β-thalassemia trait, the couple is at risk of offspring affected by intermediate forms of β-thalassemia. Nevertheless, in routine practice it is difficult, if not impossible, to predict for this risk without resorting to gene sequencing study. There is a longstanding belief that the presence of iron deficiency may spuriously lower the Hb A 2 level in β-thalassemia carriers. Recent data do not support this, but rather show that concomitant iron deficiency does not mask a diagnosis of β-thalassemia trait by Hb A 2 measurement. However, when faced with a patient with hypochromic microcytic anemia, it is always prudent to first exclude iron deficiency and find out its underlying cause, and consider a Hb pattern study if microcytosis is persistent after adequate iron replacement. The phenotype of normal HbA 2 β-thalassemia is also seen in heterozygous eγδβ-thalassemia due to the deletion of the delta-globin gene.
At the other end of the spectrum, an unusually high Hb A 2 level is associated with β-thalassemia trait caused by the uncommon β-globin gene deletions that include the promoter region. Hb A 2 may also be elevated in HIV-positive women without hypochromic microcytic indexes, those with hyperthyroidism or megaloblastic anemia, and individuals with some unstable Hbs. HPLC is the preferred method for this quantification; densitometric scanning of the Hb A 2 band on an alkaline electrophoresis gel is not recommended because of its poor precision and accuracy. In 30 to 40% of all cases of β-thalassemia minor, Hb F could be mildly elevated (1 to 2%). The life span of the RBC may be reduced, and individuals with diabetes may show a lower HbA 1c compared with normal individuals with equivalent glycemic control. The β-thalassemia mutations can be identified by direct sequencing or by multiplex PCR-based molecular testing.
δ - β -thalassemia
The δβ-thalassemias are the result of deletions that affect various parts of the β-globin locus. These deletions are partially compensated by an increased expression of the γ genes that raises the level of Hb F. The length of deletion accounts for different forms of δβ-thalassemia, including both G γ and A γ genes or only A γ, and vary from 9 to 100 kb. Hb Lepore is a hybrid of δ- and β-chains that result from a crossover between the two misaligned genes; this Hb is synthesized inefficiently and gives rise to a form of δβ-thalassemia.
Both heterozygous and homozygous conditions have been described. It is found in a variety of ethnic groups, but it is most prevalent in Eastern Mediterranean countries, especially Greece and Italy. CBC analysis of heterozygotes often shows a reduced concentration of Hb (8 to 13.5 g/dL; 80 to 135 g/L) with reduced MCV and MCH, and sometimes an increased RDW. HPLC analysis shows an Hb A peak with a normal or reduced Hb A 2 concentration and a raised Hb F concentration (between 5 and 20%), with the highest Hb F concentration seen in the Sardinian type of δβ-thalassemia.
Hereditary persistence of fetal hemoglobin
The term hereditary persistence of Hb F (HPFH) is used to describe a group of genetic conditions in which the concentration of Hb F is increased above the upper limit of the reference interval because of a reduction in β-globin synthesis and an increase in γ-globin synthesis. As for δβ-thalassemias, deletions of δ and β genes are the molecular basis for many forms of HPFH. However, the level of Hb F production in HPFH is higher than that seen in the δβ-thalassemias. Several deletional variants of HPFH have been described, including Greek, Indian, Italian, Thai, Corfu, and black. In black HPFH, the Hb F is raised to between 10 and 36% of the total Hb with normal Hb A 2 concentrations. Hb, MCV, and MCH are within the reference intervals. This condition is clinically innocuous and asymptomatic. Similarly, no clinical abnormalities are associated with Greek and Thai HPFH, although the concentration of Hb F is in the range of 15 to 25%. By mapping a variety of deletions within the β-globin gene locus that either result in δβ-thalassemia with a modest increase in Hb F and some remaining globin chain imbalance present, or HPFH with a higher Hb F level and balanced globin chain synthesis (hence normochromic normocytic red cell indices), it is found that an approximately 3-kb region upstream of the δ-globin gene is necessary for the silencing of the γ-globin genes. This region harbors binding sites of BCL11A, along with its partners, GATA-1 and HDAC1. Note that some β-thalassemia deletions, such as the Southeast Asian (Vietnamese) deletion, are associated with elevations of both Hb A 2 and Hb F (10 to 20%) in the heterozygous state.
Other HPFHs are due to point mutations in the promoter region upstream from the transcription start site in either the G γ and A γ genes, which alter the binding of one or more transcription factors; these are known as “nondeletional HPFHs.”
The increase in Hb F in nondeletional HPFH is distributed heterogeneously among the red cells (heterocellular) in otherwise normal individuals. The Hb F concentration varies between 1 and 13% of total Hb in heterozygotes and between 19 and 21% in homozygotes. No clinical or hematologic abnormalities are noted. The Hb F distribution is also heterocellular in δβ-thalassemia, while it is pancellular in deletional HPFH. Assessment of cellular Hb F distribution and enumeration of F-cells (Hb F-containing red cells) particularly in the very low range is more accurately performed by flow cytometric measurement using an anti–γ-globin chain monoclonal antibody. The quantification of Hb F level and enumeration of F-cells are also useful: (1) as a laboratory endpoint in clinical studies on the augmentation of Hb F production in sickle cell disease and β-thalassemia syndromes; and (2) as a precise hematologic marker for genetic association studies to identify candidate quantitative trait loci that are associated with enhanced γ-globin chain production and amelioration of β-globin disorders.
Hemoglobinopathies
If only single point mutations are considered, there are 1695 possible Hb variants of which 733 were identified by mid-2007. Currently, more than 900 hemoglobinopathies have been described, but only a handful show clinical significance. Recent migration from regions with a high frequency of hemoglobinopathies (Southeast Asia or Africa) to regions that had low frequencies (Western Europe, Central and South America, and Canada) has increased the incidence of hemoglobinopathies in these areas to such an extent that some western European countries have introduced neonatal testing for Hb variants. The incidental finding of a hemoglobinopathy during HPLC analysis for HbA 1c has increased both the number and the incidence of Hb variants. , Several Hb variants (e.g., Hb Rambam, Niigata, Camden) interfere with HPLC methods for quantifying HbA 1c (for a more extensive list, see Elder and colleagues ), and Hb variants have also been found as a result of interference in pulse oximetry measurements.
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