HbA2 levels in children with β-thalassemia trait associated with iron deficiency: A perspective for pediatricians

Abstract

Introduction

Thalassemia continues to pose a clinically significant global public health challenge.¹ Therefore, it is necessary to detect and prevent the disorder. In clinical practice, the critical factor for detecting β-thalassemia trait is high hemoglobin A2 (HbA2) levels (≥3.5%) on high-performance liquid chromatography (HPLC) after a suspicion finding during complete blood cell count (CBC) assessment.² Changes in erythrocyte parameters on CBC have been studied extensively and even automated using artificial intelligence,^3,4 but our clinical understanding of HbA2 changes on HPLC remains somewhat limited. Hemoglobin A2 is affected by iron deficiency, a condition even more prevalent than thalassemia. With iron deficiency, HbA2 decreases, potentially leading to a missed diagnosis of thalassemia. This established clinical practice has guided our diagnosis and management of β-thalassemia trait cases, but emerging evidence from adult studies^5-8 has suggested that this practice may not be an absolute truth. The question arises, what is the situation in pediatric cases? Could a pediatrician fail to diagnose an iron-deficient β-thalassemia carrier? Our study was designed to address this clinical question.

Methods

The participants in this study were children with a diagnosis of β-thalassemia trait followed up in the Pediatric Hematology Department of Gazi University Medical Faculty in Turkey. We assessed a cohort of 213 children comprising 106 boys and 107 girls, with a median (min-max) age of 6.0 (1.0-17.0) years.

β-Thalassemia trait was diagnosed based on red blood cell (RBC) indices on CBC and HbA2 level on HPLC, with microcytosis (mean corpuscular volume <80 fL), hypochromia (mean corpuscular hemoglobin [MCH] <25 pg/cell), and erythrocytosis (RBCs >5 × 10¹²/L) on CBC, while body iron status was normal (ferritin ≥15 ng/mL and transferrin saturation ≥15%); simultaneously increased HbA2 levels (≥3.5%) were consistent with the diagnosis. In individuals with similar results but normal HbA2 levels, the diagnosis was achieved by parental study (similar results in either parent were accepted as β-thalassemia trait).

Erythrocyte parameters of hemoglobin, hematocrit, mean corpuscular volume, MCH, MCH concentration, RBC, and RBC distribution width (RDW); status of serum iron, total iron binding capacity, transferrin saturation ([serum iron /  total iron binding capacity] × 100), and ferritin levels; and HPLC results of HbA2 and HbF levels within a specific time interval (from January 1, 2015, to December 31, 2019) were obtained from patient medical records.

Automated blood cell counts were performed using a Coulter LH 780 hematology analyzer (Beckman Coulter), biochemical parameters were analyzed using a hormone analyzer and chemiluminescent immunoassay (Immulite 1000 Immunoassay System [Siemens Healthineers]), and hemoglobin analysis was carried out using an HPLC system (VARIANT system [Bio-Rad Laboratories]).

Patients with comorbidities were not included. All patients were symptom free, and C-reactive protein was negative during testing.

Patients with iron deficiency were identified based on serum ferritin levels (<15 ng/mL); among these iron-deficient carriers, individuals exhibiting HbA2 levels of 3.5% or higher were identified. We compared the HbA2 levels of iron-deficient carriers with those of iron-sufficient individuals. After dividing the carriers into 2 groups according to their HbA2 levels as ≥3.5% and <3.5%, we examined the correlation between low HbA2 levels and ferritin. We also examined the hematologic features of individuals with low HbA2. Given the acute-phase reactant nature of ferritin, we conducted similar evaluations using transferrin saturation as the diagnostic criterion for iron deficiency (transferrin saturation <15%).

Statistical analysis was performed using SPSS, version 25.0, statistical software (IBM Corp). Data were expressed as medians (min-max). Comparisons between groups and rank correlation were performed through analysis of nonparametric tests (Mann-Whitney U test and Spearman correlation test). P < .05 was considered statistically significant.

Among the 213 children studied, 51 (23.9%) were found to have iron deficiency. In 28 of these 51 iron-deficient carriers (54.9%), HbA2 levels were 3.5% or higher FIGURE 1. The median HbA2 value among iron-deficient carriers was 4.1%, which was lower than among individuals without iron deficiency but still within the diagnostic threshold (median, 4.1% [SD, 1.5%] vs. 4.9% [SD, 1.4%], P < .007] TABLE 1. Among the 213 children, 67 (31.5%) exhibited HbA2 levels below 3.5%; in these cases, low HbA2 levels did not show a significant correlation with ferritin (0.226).

FIGURE 1

The distribution of the iron-deficient carriers presenting with HbA2 levels of 3.5% or more is shown as a bar graph (red bars). HbA2 <3.5%: n = 23; HbA2 ≥3.5%: n = 28. HbA2, hemoglobin A2.

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Similar results were obtained when transferrin saturation was used; 55 of 213 (25.8%) children had iron deficiency, and 30 (54.5%) of these iron-deficient carriers had HbA2 levels still within the diagnostic range (median, 4.1% [SD, 1.5%]). No correlation was found between low HbA2 levels and transferrin saturation in the 67 children with low HbA2 levels (0.280).

Even within the group of iron-sufficient individuals, a portion exhibited low HbA2 levels. In other words, among 67 individuals with low HbA2 levels, there were patients without iron deficiency (26.9% based on ferritin and 26.6% based on transferrin saturation) FIGURE 2. TABLE 2 shows the hematologic results of these cases.

FIGURE 2

The distribution of the iron-sufficient carriers presenting with HbA2 levels below 3.5% is shown as a bar graph (red bars). HbA2 <3.5%: n = 43; HbA2 ≥3.5%: n = 117. HbA2, hemoglobin A2.

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Iron deficiency is a common companion of β-thalassemia trait.⁹ In iron deficiency, the normal preference for HbA formation is further exaggerated, leading to a reduction in HbA2.¹⁰ Therefore, in cases of β-thalassemia trait accompanied by iron deficiency, low HbA2 levels are often attributed to iron deficiency, although caution is warranted in such cases because the correlation between low ferritin levels and decreased HbA2 levels is observed in cases of pure iron deficiency, not in individuals with β-thalassemia trait.¹⁰ Correspondingly, in cases of β-thalassemia trait, multiple linear regression analysis has demonstrated a significant association between low HbA2 levels and β-thalassemia variations, independent of low ferritin levels (P < .001).^7,11 These variations primarily involve abnormalities in the δ-globin gene (concomitant inheritance of variant δ-globin chains [δβ-thalassemia trait], δ-thalassemia variations, and rare deletions involving the δ-globin gene, with decreasing frequency).^12,13 Therefore, it would not be correct to attribute low HbA2 levels to iron deficiency without studying variation analysis. Indeed, an increasing amount of evidence indicates that individuals with iron deficiency and β-thalassemia trait consistently exhibit elevated HbA2 levels^5-8 because in individuals with β-thalassemia trait, the availability of β chains is also decreased; therefore, accompanied iron deficiency will not disproportionately reduce the relative proportion of HbA2.¹⁰

In our study, cases with HbA2 levels below 3.5% presented a diagnostic challenge because they could represent classic β-thalassemia trait with decreased HbA2 levels due to iron deficiency, HbA2-normal β-thalassemia trait, or δβ-thalassemia trait, regardless of iron status. Because the potential impact of iron treatment is unknown and the variation analysis was not available at the time of evaluation, a systematic approach was proposed to aid in differentiation. Patients with accompanying iron deficiency and low HbA2 levels lacked distinction among these conditions because all 3 scenarios could occur concurrently. In patients without accompanying iron deficiency, however, the possibilities narrowed to either HbA2-normal β-thalassemia trait or δβ-thalassemia trait. The primary clinical focus remained on identifying β-thalassemia trait rather than on defining its subgroups. Nonetheless, discrimination between these conditions was facilitated by assessing RDW on CBC analysis and HbF levels in HPLC. Elevated RDW coupled with high HbF levels favored deltaβ-thalassemia trait, indicating a potential role for these parameters in distinguishing between HbA2-normal β-thalassemia trait and δβ-thalassemia trait. Notably, RDW emerged as the most effective parameter for discriminating between δβ-thalassemia trait and β-thalassemia trait, with the degree of anisocytosis correlating strongly with HbF levels.^14,15 Thus, a comprehensive evaluation of RDW and HbF levels, without undertaking additional tests, contributed significantly to the distinction between these thalassemia subtypes.

In our cohort, among the 67 patients with HbA2 levels below 3.5%, the RDW and HbF results of 43 patients without iron deficiency (ferritin ≥15 ng/mL) remained consistent (median, 16.1% [SD, 3.0%] and 0.8% [1.5%], respectively), aligning with the characteristics of HbA2-normal β-thalassemia trait. Similar concordant results were observed when transferrin saturation was used as a criterion for iron deficiency (transferrin saturation ≥15%) (median, 16.0% [2.9%] and 0.8% [0.8%], respectively).

In our study, which mirrors real-world clinical practice, HbA2 levels were 3.5% or higher in the majority of iron-deficient carriers, with a median value of 4.1%. This finding underscores the reliability of HbA2 as a diagnostic tool for β-thalassemia trait screening, even in the presence of iron deficiency (ineffectiveness of iron deficiency in β-thalassemia trait). In addition, within the group of carriers without iron deficiency, a subset exhibited low HbA2 levels (effect of underlying thalassemia variation). The goal of a dependable screening test is to minimize false negatives while accepting a minimal rate of false positives.⁴ In the context of HbA2-based identification of β-thalassemia trait, the complete elimination of false negatives is not feasible, irrespective of the presence of accompanying iron deficiency.

In conclusion, drawing from a synthesis of the existing literature on molecularly confirmed β-thalassemia carriers and the findings of our study, we advocate the following recommendations to pediatricians in cases where genetic testing is not available: In suspected cases of β-thalassemia trait, assess iron parameters and HbA2 levels concurrently. In cases with no iron deficiency and a high HbA2 level, the diagnosis becomes straightforward. If iron deficiency is present and HbA2 levels are found to be below 3.5%, continue the evaluation for a family study without distraction. Low HbA2 levels in these cases may be related to the underlying thalassemia variation rather than iron deficiency because iron deficiency does not significantly reduce HbA2 levels in β-thalassemia trait.

Conflict of interest disclosure: The authors have nothing to disclose.

References