Hemoglobin A₂ (HbA₂) is a minor adult hemoglobin variant composed of two α-globin chains and two δ-globin chains (α₂δ₂), with each chain bound to a heme group, structurally analogous to the predominant hemoglobin A (HbA; α₂β₂) but differing in the β-like δ chains.[1] It constitutes 2.0–3.3% of total hemoglobin in healthy adults, a level that stabilizes after infancy as fetal hemoglobin declines.[2] This tetrameric protein is encoded by the HBA1/HBA2 and HBD genes on chromosomes 16 and 11, respectively.[3]The primary function of HbA₂ mirrors that of other hemoglobins: reversible binding and transport of oxygen from the lungs to tissues and carbon dioxide from tissues to the lungs, facilitated by its normal oxygen affinity and cooperative binding properties.[1] Due to its low concentration, however, HbA₂ contributes minimally to overall oxygen delivery in normal physiology, and no unique physiological role has been definitively established beyond this.[4] Notably, in sickle cell anemia, HbA₂ inhibits the polymerization of deoxygenated hemoglobin S (HbS), potentially reducing the severity of vaso-occlusive events by interfering with HbS fiber formation.[3]Clinically, HbA₂ holds significant diagnostic value, particularly in hemoglobinopathies; its levels rise to 4–8% in β-thalassemia carriers due to compensatory δ-globin production amid reduced β-globin synthesis, making it a cornerstone for trait detection via hemoglobin electrophoresis or high-performance liquid chromatography (HPLC).[5] Reduced HbA₂ (<2.0%) signals δ-thalassemia or structural δ-globin variants, which can mask β-thalassemia and necessitate molecular confirmation.[2] Variants of HbA₂, such as those altering δ-chain stability or expression, occur at low frequencies (e.g., 1–2% in some populations) and may complicate routine screening but rarely cause overt disease.[2] Emerging research explores elevating HbA₂ as a therapeutic strategy for sickle cell disease, leveraging its anti-polymerization effects, though challenges like reciprocal regulation with fetal hemoglobin persist.[4]
Overview
Molecular Structure
Hemoglobin A2 (HbA₂) is a tetrameric protein composed of two identical α-globin chains and two identical δ-globin chains, forming the quaternary structure α₂δ₂, with an approximate molecular weight of 64,500 Da. This structure enables it to function as an oxygen carrier, similar to the predominant adult hemoglobin variant HbA (α₂β₂). Each of the four polypeptide chains folds into a globular domain with eight α-helices (labeled A through H), characteristic of the globin fold family.[6]The δ-globin chain in HbA₂ exhibits structural similarities to the β-globin chain in HbA, including conserved heme-binding pockets that accommodate a protoporphyrin IX ring with a central ferrous iron (Fe²⁺) atom. The iron is axially coordinated by the imidazole nitrogen of a proximal histidine residue at position F8 (His87 in α, His92 in δ) and, upon oxygenation, interacts with a distal histidine at E7 (His58 in α, His63 in δ) to stabilize the bound oxygen molecule without irreversible oxidation to ferric iron. However, the δ-chain differs from the β-chain by 10 specific amino acid substitutions out of 147 residues, primarily at positions such as 2 (His→Asn), 116 (His→Arg), and 122 (Glu→Gln), which introduce subtle variations in surface charge and hydrogen bonding patterns.[7]These substitutions lead to minor conformational adjustments in the δ-subunits, particularly at the α₁δ₂ and α₂δ₁ interfaces, enhancing intersubunit interactions and contributing to the tetramer's slightly higher thermal stability compared to HbA. X-ray crystallographic studies have elucidated the structure of HbA₂ at 2.20 Å resolution in the relaxed (R₂) state, revealing these δ-specific changes that promote tighter tetramer assembly and a modestly increased oxygen affinity relative to HbA. In adults, HbA₂ normally accounts for 2-3% of total circulating hemoglobin.[6][8][9]
Biosynthesis and Genetic Basis
Hemoglobin A2 (HbA2) is composed of two alpha-globin chains and two delta-globin chains, encoded by the HBD gene located on the short arm of chromosome 11 at position 11p15.4. The HBD gene spans approximately 1.6 kb and consists of three exons, which encode the 147-amino-acid delta-globin protein. The delta-globin chain exhibits 93% sequence identity with the beta-globin chain, differing by only 10 amino acids, a high degree of homology that underscores their shared evolutionary origin within the beta-globin gene cluster.[7]Transcription of the HBD gene is regulated by the locus control region (LCR), a powerful enhancer element located approximately 50 kb upstream of the beta-globin gene cluster on chromosome 11.[10] The LCR facilitates long-range chromatin interactions that promote high-level, tissue-specific expression of globin genes in erythroid cells.[10] Key transcription factors, including GATA1 and KLF1 (also known as EKLF), bind to the LCR and promoter regions to activate HBD transcription, with KLF1 playing a central role in adult erythroid differentiation by directly influencing delta-globin expression levels.[11] These factors ensure coordinated switching from fetal to adult hemoglobin production, though HBD expression remains low compared to the adjacent HBB gene encoding beta-globin.[11]At the post-transcriptional level, HbA2 biosynthesis is limited by the reduced stability of delta-globin mRNA relative to beta-globin mRNA, which contributes to lower translation efficiency and overall protein output.[12] Studies have shown that delta-globin mRNA has a shorter half-life, influenced by differences in 3' untranslated region (UTR) sequences and associated stability factors, leading to decreased availability for ribosomal translation in erythroid precursors.[13] This mechanism helps maintain the typical 2-3% proportion of HbA2 in adult hemoglobin, preventing competition with the dominant HbA.[13]The HBD gene demonstrates strong evolutionary conservation across primates, with evidence of purifying selection maintaining its sequence integrity over millions of years, despite its minor expression in adults.[14] This conservation suggests the delta-globin gene persists as a duplicated remnant within the beta-globin cluster, which arose from ancient gene duplications involving ancestral globin loci that also gave rise to fetal gamma-globin genes.[14] Such evolutionary retention may provide a genetic reservoir for adaptive responses, as seen in certain hemoglobinopathies where delta-globin compensates for beta-globin defects.[7]
Physiological Role
Contribution to Oxygen Transport
Hemoglobin A2 (HbA₂), comprising 2-3% of total hemoglobin in adult red blood cells, binds oxygen reversibly through the iron in its heme groups, much like the predominant HbA (α₂β₂). Its oxygen-binding properties exhibit a slightly higher affinity than HbA, reflected in a p50 value of approximately 24 mmHg compared to 26 mmHg for HbA, facilitating marginally enhanced oxygen uptake under physiological conditions. This cooperative binding follows a sigmoidal dissociation curve, enabling efficient oxygen loading in the lungs and release in tissues, though its low abundance limits its overall impact on systemic oxygen transport.The tetrameric structure of HbA₂ (α₂δ₂) contributes to tetramer stability within red blood cells, similar to HbA, reducing the likelihood of dissociation into dimers under physiological shear and osmotic stresses.[15] This enhanced stability helps maintain functional hemoglobin integrity during circulation. Regarding allosteric regulation, HbA₂ displays a diminished alkaline Bohr effect, approximately 50% that of HbA, attributable to fewer key histidine residues in the δ chains (e.g., Ser143 instead of His143 in β chains) that participate in proton binding and pH-dependent oxygen affinity modulation.Interaction with 2,3-bisphosphoglycerate (2,3-BPG) is notably reduced in HbA₂ due to alterations in the δ chain binding pocket, including the substitution at position 143, resulting in minimal modulation of oxygen affinity compared to the pronounced effect observed in HbA. Consequently, HbA₂ contributes less to the rightward shift of the oxygen dissociation curve in response to tissue hypoxia or increased 2,3-BPG levels. In scenarios such as β-thalassemia carriers, where β-globin synthesis is impaired, the presence of HbA₂ provides minimal compensatory support for oxygen delivery by substituting for reduced HbA function, helping to preserve near-normal tissue oxygenation despite its minor proportion. Overall, while HbA₂ plays a supportive role in oxygen transport, its contributions are subtle and primarily structural rather than dominant physiological.
Expression Across Life Stages
Hemoglobin A2 (HbA2) expression is minimal during fetal development, typically constituting less than 0.5% of total hemoglobin, with levels often reported as 0% in cord blood samples due to the predominance of fetal hemoglobin (HbF).[16] This low expression reflects the developmental prioritization of gamma-globin synthesis over delta-globin (HBD gene product) within the beta-globin gene cluster. Postnatally, as the gamma-to-beta globin switch occurs, HbA2 levels begin to rise, coinciding with the decline in HbF and the dominance of beta-globin production. In newborns, there is a transient increase in HbA2 during this transition period, driven by the activation of adult hemoglobin synthesis pathways.[17]By 6-12 months of age, HbA2 levels reach the adult plateau of approximately 2-3% of total hemoglobin, stabilizing thereafter in healthy individuals.[16][18] This maturation aligns with the completion of the globin switch, where HbA2 assumes its minor role alongside HbA. Baseline HbA2 expression remains tightly regulated within the beta-globin locus, ensuring consistent proportions in adults under normal conditions. However, hormonal factors can influence expression during physiological stress, such as hypoxia or anemia, by modulating overall erythropoiesis, though specific impacts on HbA2 are secondary to broader red cell production dynamics.Rare genetic variants, such as those associated with hereditary persistence of fetal hemoglobin (HPFH), can alter the timing and extent of HbA2 expression; for instance, deletions or mutations overlapping the delta gene region in δβ-thalassemia or HPFH often result in reduced or absent HbA2 alongside persistent HbF elevation into adulthood.[19][20] These variants highlight the interconnected regulation within the beta-globin cluster, where disruptions can delay the full transition to adult hemoglobin profiles.
Clinical Relevance
Elevated HbA2 Levels
Elevated levels of hemoglobin A2 (HbA2), typically exceeding 3.5% of total hemoglobin, are most commonly associated with β-thalassemia trait, a heterozygous condition resulting from mutations in the HBB gene that impair β-globin chain synthesis. In affected individuals, HbA2 often ranges from 4% to 8%, serving as a key indicator of this carrier state. This elevation arises because the reduced availability of β-globin chains disrupts the formation of predominant HbA (α₂β₂), leading to a compensatory pairing of excess α-chains with δ-chains to form more HbA2 (α₂δ₂).[5]The mechanism involves a relative increase in HbA2 proportion rather than an absolute overproduction of δ-globin chains. Mutations, particularly those affecting the β-globin promoter, reduce competition for the locus control region (LCR) upstream in the β-globin gene cluster, allowing enhanced interaction of the LCR with the adjacent δ-globin (HBD) gene and promoting its transcription. As a result, δ-globin expression rises modestly to balance the globin chain imbalance, but total δ-chain synthesis remains limited, with HbA2 rarely surpassing 10% even in carriers.[21]Mild elevations in HbA2 can also occur in non-thalassemic conditions, including megaloblastic anemias from vitamin B12 or folate deficiency, where impaired DNA synthesis in erythroid precursors favors δ-chain production, raising HbA2 to around 3.5-4.5%; hyperthyroidism, due to thyroid hormone stimulation of δ-globin expression; and antiretroviral therapies, particularly zidovudine, which alter globin synthesis in immature red cells. Rare genetic causes include duplications or activating variants in the δ-globin gene that boost its output, leading to HbA2 levels up to 6-10% without β-chain defects. Notably, iron deficiency anemia generally lowers HbA2 rather than elevating it, potentially masking underlying thalassemia.[22][5]Clinically, elevated HbA2 in β-thalassemia carriers is often asymptomatic, though mild microcytic hypochromic anemia may be present and discovered incidentally during routine blood tests. Detection of levels above 4% signals the need for genetic counseling to evaluate inheritance risks, as offspring of two carriers face a 25% chance of β-thalassemia major. Such findings prompt targeted screening, including family studies and molecular testing, to prevent severe disease in future generations.[5][23]
Reduced HbA2 Levels
Reduced hemoglobin A2 (HbA2) levels, typically below the normal adult range of 2-3.5%, are most commonly associated with δ-thalassemia, a condition arising from mutations in the HBD gene on chromosome 11 that encode the δ-globin chain.[24] In heterozygous δ-thalassemia, a single mutated allele leads to mildly reduced δ-globin synthesis, resulting in HbA2 comprising less than 2% of total hemoglobin, while homozygous forms cause even more pronounced reductions due to absent or severely impaired δ-globin production.[25] These mutations often involve point substitutions, such as those affecting splicing or promoter regions, or large deletions that disrupt HBD transcription, directly lowering δ-chain mRNA levels or destabilizing the resulting protein.[26]Other etiologies include α-thalassemia, where reduced α-globin availability creates an imbalance in globin chain assembly, mildly suppressing HbA2 formation as fewer α-chains pair with δ-chains to form functional tetramers.[27] Folate deficiency in β-thalassemia carriers similarly contributes by disrupting DNA synthesis in erythroid precursors, leading to suppressed HbA2 expression without altering vitamin B12 status.[24]The underlying mechanisms generally involve direct curtailment of δ-globin synthesis or secondary effects from chain imbalance, with no typical compensatory upregulation of β-globin to offset the deficit.[28] Clinically, isolated reduced HbA2 from δ-thalassemia is often asymptomatic, presenting only as subtle microcytosis on blood smears without significant anemia or organ involvement. However, in compound heterozygotes carrying both δ-thalassemia and β-thalassemia alleles, the condition manifests as a milder microcytic anemia compared to β-thalassemia trait alone, with attenuated red cell abnormalities due to partially preserved hemoglobin variants.
Role in Thalassemia Diagnosis
Hemoglobin A2 (HbA2) measurement serves as a cornerstone in the diagnosis of β-thalassemia carriers, where levels exceeding 3.5%—typically ranging from 3.5% to 7%—combined with microcytosis (mean corpuscular volume [MCV] <80 fL) and normal iron studies (e.g., normal serum ferritin and transferrin saturation) strongly indicate the trait.[29][30] This elevation arises due to the relative increase in δ-globin chain production compensating for reduced β-globin synthesis, distinguishing β-thalassemia trait from other causes of microcytosis like iron deficiency anemia, where HbA2 levels are usually normal or reduced.[31][27]In contrast, δ-thalassemia is characterized by HbA2 levels below 2%, often presenting as a silent carrier state with minimal hematological abnormalities unless co-inherited with β-thalassemia, in which case it may result in normal HbA levels but mild anemia and elevated fetal hemoglobin (HbF).[2][32] This reduction reflects impaired δ-globin production, and diagnosis typically requires awareness of this pattern in individuals with unexplained low HbA2 alongside microcytosis.[33]The diagnostic algorithm for thalassemia begins with HbA2 quantification via high-performance liquid chromatography (HPLC) or capillary electrophoresis as a first-line screening tool in at-risk populations, followed by molecular genetic testing such as sequencing of the HBB (β-globin) and HBD (δ-globin) genes to confirm mutations when phenotypic findings are suggestive.[34][35] This approach effectively differentiates thalassemia from iron deficiency by integrating HbA2 results with iron status assessments, as concurrent iron deficiency can artifactually lower HbA2 in β-thalassemia carriers, necessitating iron repletion prior to retesting if indicated. Similarly, folate deficiency in β-thalassemia carriers can lower HbA2 levels, and supplementation may be needed for accurate assessment.[36][31][24]Population-specific considerations are essential, as HbA2 cutoffs may require adjustment in high-prevalence regions; for instance, carriers in Mediterranean and Southeast Asian populations often exhibit slightly higher baseline HbA2 levels (mean around 4.5-5%), potentially necessitating cutoffs above 3.5% to optimize specificity.[37][38] Additionally, false positives can occur in HbE trait, prevalent in Southeast Asian groups, where HbE co-elutes with HbA2 in chromatographic methods, inflating apparent HbA2 readings above 3.5% and mimicking β-thalassemia; confirmatory hemoglobin variant analysis is thus recommended in such cases.[39][40]
Measurement and Analysis
Laboratory Detection Methods
High-performance liquid chromatography (HPLC) serves as the gold standard for quantifying hemoglobin A2 (HbA2) due to its high precision and automation capabilities.[41] This method employs weak cation-exchange chromatography, where hemoglobin variants are separated based on charge differences as the ionic strength of the mobile phase increases during elution.[42] In the procedure, a red blood cell lysate is injected into the system, and absorbance is monitored at 415 nm and 690 nm to detect peaks; HbA2 typically elutes with a retention time of approximately 3.3 to 3.9 minutes, depending on the instrument, and is quantified as a percentage of total hemoglobin with a precision of ±0.1% (standard deviation ≤0.05%, coefficient of variation ≤2%).[43] Calibration using the World Health Organization international reference reagent (HbA2 at 5.3 ± 0.066%) ensures accuracy, and systems must demonstrate complete baseline separation between HbA and HbA2 peaks.[41]Capillary electrophoresis (CE) provides an automated alternative for HbA2 measurement, particularly effective for detecting low levels in neonatal samples.[44] The principle involves separation by charge and size within a silica capillary under high voltage (10-30 kV), utilizing electro-osmotic flow to migrate hemoglobin zones, with detection at 415 nm.[42] Procedure-wise, a small volume of hemolysate is injected, and HbA2 is quantified via peak area analysis, achieving precision comparable to HPLC at ±0.1%; it excels in newborn screening by resolving HbA2 from high fetal hemoglobin backgrounds in dried blood spots or fresh samples.[41][44] HbA2 migrates separately in an order akin to alkaline electrophoresis, though co-migration with variants like HbE or Hb O-Arab can occur.[42]Cellulose acetate electrophoresis represents an older, manual method that remains cost-effective for HbA2 visualization in resource-limited settings.[45] It operates on the principle of charge-based separation on a cellulose acetate strip at alkaline pH (8.2-8.6), where hemoglobin bands are formed and subsequently stained or eluted for densitometric quantification.[46] The procedure requires preparing a hemolysate from 1-2 mL of washed red cells, applying it to the strip, running the electrophoresis, and measuring absorbance at 415 nm after elution, yielding a precision of ±0.1% with experienced operators but generally less quantitative than automated techniques (relative standard deviation ~2.0%).[42][41] Bands are visualized under these conditions, with HbA2 migrating near HbC and HbE.[42]Common limitations across these methods include interference from hemoglobin variants such as HbC, HbE, or HbS, which may co-elute or co-migrate with HbA2, potentially leading to inaccurate quantification; for instance, HbC and HbE overlap in cellulose acetate and some HPLC/CE systems.[42] Samples typically require collection in K2-EDTA anticoagulant, with whole blood or lysates stable for up to 2-3 weeks when refrigerated at 4°C, though freezing at -20°C extends viability to 1 month.[42] Interlaboratory variability in HPLC can reach 6.0-9.6%, underscoring the need for standardized reference materials and quality control.[42]
Clinical Interpretation Guidelines
The normal reference range for HbA2 in adults is typically 2.0-3.5% of total hemoglobin, though slight variations exist across laboratories and populations.[47] Reference ranges may require adjustment for ethnicity, as studies in diverse groups such as those of Chinese descent report narrower intervals like 2.3-3.1%, emphasizing the need for population-specific norms to avoid misinterpretation in carrier screening.[48]Levels below 1.5-2.0% often indicate δ-thalassemia trait, where reduced δ-globin synthesis lowers HbA2 production, potentially masking co-inherited β-thalassemia if not considered.[49] Conversely, HbA2 exceeding 3.5% strongly suggests β-thalassemia trait, with values typically rising to 3.5-7.0% due to compensatory δ-globin expression in the presence of β-globin defects.[50] Borderline elevations (3.1-3.5%) warrant further evaluation, as they may reflect mild β-thalassemia variants or modifiers like α-thalassemia co-inheritance.[51]Clinical interpretation of HbA2 results should integrate with complete blood count (CBC) parameters, particularly mean corpuscular volume (MCV <80 fL) and mean corpuscular hemoglobin (MCH <27 pg), which signal microcytic anemia suggestive of thalassemia traits when combined with elevated HbA2.[30] Hemoglobin electrophoresis or high-performance liquid chromatography (HPLC) for HbF quantification complements this, as elevated HbF (>2%) alongside high HbA2 supports β-thalassemia diagnosis, while isolated low MCV with normal HbA2 may point to α-thalassemia or iron deficiency.[52]In prenatal screening for high-risk populations (e.g., Mediterranean, Southeast Asian, or South Asian descent), an algorithm begins with CBC to identify low MCV, followed by HbA2 measurement; if HbA2 >3.5%, confirm β-thalassemia carrier status via genetic testing, and offer partner screening to assess compound heterozygote risk (25% per pregnancy).[53] If both parents are carriers, proceed to chorionic villus sampling or amniocentesis for fetal genotyping, prioritizing early gestation to inform options.[54]As of 2025, World Health Organization (WHO) guidelines endorse HPLC-based HbA2 quantification as the primary method for β-thalassemia carrier detection due to its accuracy and accessibility in resource-limited settings.[55] For ambiguous cases, such as borderline HbA2 or silent carriers, next-generation sequencing (NGS) of HBB and related genes is recommended for confirmation, improving detection rates in diverse populations.[56]Common pitfalls in HbA2 interpretation include transient alterations during pregnancy, where levels may decrease slightly (e.g., by 0.2-0.5%) due to hemodilution or iron demands, potentially underestimating β-thalassemia trait; retesting postpartum is advised.[57] Recent blood transfusions can dilute endogenous HbA2 with donor hemoglobin (typically 2-3%), falsely normalizing elevated levels in thalassemia patients; interpretation should thus exclude samples collected within 3-4 months post-transfusion, with retesting after resolution.[58]