Sinlapamongkolkul

An estimated 300-million children worldwide had anemia in 2011,[1] and iron deficiency anemia (IDA) remains the most common cause of this to date. The World Health Organization (WHO) publishes an international anemia control guideline that states all children and women living in settings where the prevalence of anemia exceeds 20% should receive supplemental iron.[2] In Thailand, this recommendation has been adopted by the Department of Health, Ministry of Public Health, which recommends a universal iron supplement for Thai infants over six months old to prevent IDA when these babies come for routine vaccination. Iron supplementation continues until 24 months of age with 12.5 mg of elemental iron weekly, according to WHO Guideline (2011).[3,4]
Compared to placebos or no intervention, intermittent iron supplementation is considered effective in reducing the risk of anemia or iron deficiency (ID) in children younger than 12 years old.[5] This is because infants older than six months have a high prevalence of IDA, which can impair physical, behavioral, and cognitive functions and result in persistent neurocognitive defects, despite receiving iron therapy later.[6] However, local Thai practitioners, particularly pediatricians, have concerns about this policy since there is a high prevalence of thalassemia and hemoglobin disorders in the country.[7] It is widely accepted that thalassemia disease could significantly increase the risk of iron overload, leading to iron toxicity later in life.[8-9]
Thalassemia is characterized by inherited mutations of α and β globin genes causing decreased globin synthesis. At least 5.2% of the world population carries one allele of globin gene variants (carrier or trait).[10] For α-thalassemia, there are two types based on molecular defects: α⁰-thalalassemia caused by deletions of two linked α-globin genes in cis (--/αα) and α⁺-thalassemia caused by deletions of one α-globin gene (-α/αα) or nucleotide mutations (α^Tα/αα or αα/αα^T). Coinheritance of two affected alleles in autosomal recessive mode leads to chronic hemolytic anemia and ineffective erythropoiesis, known as thalassemia disease.[11] On the other hand, hemoglobinopathy is mainly caused by mutations of coding sequences and produces qualitative defects. Several hemoglobinopathies are innocuous and do not lead to any clinical consequences.[12] However, some mutations such as hemoglobin E (Hb E) at codon 26 of the β-globin genes (GAG>AAG) also have quantitative effects, and an interaction of Hb E with β-thalassemia mutations results in Hb E/β-thalassemia syndrome with heterogeneous clinical severity.
Around 30 to 40 percent of Thais are thalassemia carriers, including α-thalassemia, β-thalassemia, and Hb E. Due to a high prevalence of all genotypes, it is not uncommon to find individuals with combined α and β-globin abnormalities.[13-16] Collectively, these thalassemia traits, simple or in combination, are asymptomatic and do not require specific treatment; these are classified as “thalassemia minor”. Individuals with homozygous Hb E (Hb E/E) carrying two defective β-globin genes also present with milder forms of anemia without hepatosplenomegaly or blood transfusion being required.[17]
Several previous studies have examined iron status in patients with thalassemia,[18-21] but little is known about thalassemia minor in comparison to normal populations,[22] particularly in infants. Iron overload is one of the most common complications in those with thalassemia due to blood transfusions and increased iron absorption.[8-9] Intestinal iron intake in thalassemia is usually enhanced due to hepcidin suppression by the upregulation of erythropoietic markers such as GDF-11, GDF-15 and Erfe in response to chronic anemia and erythropoietin drive.[23-25] Hepcidin controls iron intake through duodenal enterocytes by limiting the expression of ferroportin: an intestinal iron gateway into circulation. Research consistently shows hepcidin suppression in thalassemia patients.[26,27] Recently, a Sri Lankan study demonstrated that β-thalassemia carriers had mildly suppressed hepcidin concentrations out of proportion to their iron stores. It has been suggested that a widespread distribution of iron supplementation could possibly increase the risk of harmful iron overload in β-thalassemia carriers.[28] In Thailand, there has been no data on iron status and hepcidin levels in the pediatric population with our common thalassemia traits of α-thalassemia and Hb E and homozygous HbE, especially in infants who receive supplements through our national program.
Our main objective was to determine the iron status in infants aged six to 12 months from our Well Baby Clinic and identify the prevalence of ID and IDA among those with or without thalassemia. In addition, we evaluated clinical and laboratory characteristics of both groups to identify which factors, including hepcidin levels, would significantly influence iron status. We aimed to illustrate whether infants with thalassemia are at similar or lower risk of ID or IDA as compared to the similarly-aged general population and, thus, supply evidence regarding safe universal iron supplementation for Thai infants.

Study population. This is a cross-sectional descriptive study approved by the Human Research Ethics Committee of Thammasat University (Medicine) (MTU-EC-PE-2-006/59). From June 2016 to June 2017, six- to 12-month-old infants attending the Well Baby Clinic at Thammasat University Hospital for vaccinations and scheduled checkups were randomly recruited. Written informed consent was given by their parents or legal guardians. Our inclusion criteria were term newborns (38-42 weeks gestation) with a birthweight between 2,500-4,000 grams having no prenatal and perinatal complications such as severe birth asphyxia, severe respiratory distress, or neonatal intensive care unit admission. Infants with chromosome abnormalities/syndromes, infectious/inflammatory diseases and any acute health problems were excluded. All clinical samples were collected before routine universal iron supplementation. We obtained demographic/clinical data through direct interviews with two investigators (PSi and PSu). Weight and length of participants were measured and evaluated by Z-score, according to WHO guidelines.[29] The Z-scores of weight-for-lengths below or above two standard deviations (SD) are categorized as underweight and overweight, respectively.
Hematological and biochemical evaluation. All participants underwent complete blood count evaluation using automated cell count (UniCel®DxH 800, Beckman Coulter, Brea, USA), hemoglobin typing by automated capillary electrophoresis analyzer (MINICAP, Sebia, Lisses, France), iron parameters including serum iron (SI), total iron binding capacity (TIBC) using a fully automated quantitative assay, and serum ferritin by electrochemiluminescence immunoassay (ECLIA or Elecsys® technology, Roche Diagnostics, Penzberg, Germany). SI, TIBC and serum ferritin assays were performed using a ROCHE COBAS BIO centrifugal analyzer according to manufacturer’s instructions.[30]
Hepcidin, a cysteine-rich 25-amino acid peptide hormone, is produced from 84-amino acid pro-hepcidin in the liver and secreted into blood circulation, functioning iron homeostasis and anti-microbial activity. Accordingly, methods have been developed for identification and quantitation of pro-hepcidin and hepcidin in blood and urine specimens, including high-performance liquid chromatography/electrospray ionization-mass spectrometry (HPLC/ESI-MS), enzyme-linked immunosorbent assay (ELISA) methods. The HPLC/ESI-MS method is sensitive and selective; nonetheless, it requires a very expensive sophisticated instrument system and a highly experienced analyst.[31,32] In comparison, the ELISA, including competitive- and sandwich types, require specific antibody/antibodies against hepcidin epitopes, sensitive colorimetric /chemiluminescent reactions (rank of ng/mL and pg/mL), and commercially available kits by many manufacturers. In this study, we used a competitive ELISA kit (Product number CEB979Hu, Cloud-Clone Corporation, Wuhan, PR China), with afternoon blood sampling to prevent diurnal variations,[33,34] for determination of serum hepcidin, of which the quality control shows very good sensitivity (low limit of detection <11 pg/mL), specificity (no significant cross reactivity between hepcidin and analogues), recovery (82-90%), precision (coefficient of variation <10% for intra-assay and <12% for inter-assay) and linearity (86-102% for diluted serum samples).
Molecular analysis. Genomic DNA was extracted from peripheral blood leukocytes using a standard protocol of phenol-chloroform extraction. Alpha-globin genotyping was performed by a single-tube multiplex gap polymerase chain reaction (Gap-PCR) for detecting seven common α-globin deletions (--^SEA, --^THAI, -(α) ^20.5, --^FIL, -- ^MED-α^3.7, -α^4.2) and a single-tube multiplex amplification refractory mutation system (ARMS-PCR) for screening six common non-deletional α-globin mutations in Thailand: initiation codon (ATG➝A-G), codon 30 (ΔGAG), codon 59 (GGC➝GAC), codon 125 (CTG➝CCG) or Hb QuangSze, termination codon (TAA➝CAA) or Hb Constant Spring, and termination codon (TAA➝TAT) or Hb Paksé.[35] Beta-globin genotyping was performed by ARMS-PCR for detecting 16 common beta-globin mutations (-28, CD8/9, CD17, CD19, CD26 (Hb E), CD26 G>T (stop codon), CD27/28, IVSI-I, IVSI-5, CD35, CD41, CD41/42, CD43, CD71/72, CD95 and IVSII-654).[36] A single-tube multiplex Gap-PCR and enzymatic amplification was used for common beta-globin gene deletions (3.48 kb, 619 bp, Filipino (β)°, SEA HPFH (β)°, Chinese ^Gγ( ^Aγδβ)°, Thai (δβ)°,Hb Lepore, HPFH-6 ^Gγ( ^Aγδβ)°, Siriraj-thal ^Gγ( ^Aγδβ)°, Asian Indian type A, and Asian Indian type B).[37,38] Hemoglobin E testing was studied by restriction fragment length polymorphism (RFLP)-PCR utilizing MnlI restriction enzyme.[39]
Definitions. Hemoglobin (Hb) <11 g/dL was used to define anemia, in line with WHO criteria.[40] Participants were classified as having ID if their serum ferritin (SF) was <30 ng/mL or transferrin saturation (TS) was <16% (TS = SI/TIBC x100).[41] IDA was diagnosed if there was compatibility with either laboratory criteria or a therapeutic response to oral iron therapy (syrup of ferric hydroxide polymaltose complex 3-6 mg/kg/day for 8-12 weeks, as prescribed) and followed up by hematologists. The coexisting causes and possible risk factors of ID need to be further identified, especially unresponsiveness to oral iron therapy. The diagnosis of β-thalassemia trait was based on Hb A2 level >3.5%. Infants with Hb F >10% were investigated for common beta-globin gene deletions to diagnose δβ-thalassemia traits and hereditary persistence of fetal hemoglobin (HPFH), as described in previous studies.[37,42]
Statistical analysis. The sample size was calculated from this formula: N = Z²pq/d², Z = 1.96, p = 0.4, q = 0.6, d = 0.07. The study population, according to the prevalence of thalassemia in Thailand (30-40%),[13,15] was 188; we approximated needing 200 participants, reflecting a 10% dropout. Demographic data was summarized as frequency and percentage for qualitative data and as mean and SD for quantitative data. Student’s t-test or Mann-Whitney U test was used to compare continuous variables; the chi-square or Fisher’s exact test was used for categorical variables, as appropriate. Univariate and multivariate logistic regression analyses were performed to identify risk factors: P of < 0.05 was considered statistically significant.

Clinical characteristics. A total of 206 infants, with 114 males (55.3%) and a mean age of 8.2 months (SD 2.0, range 6-12 months), were randomly enrolled. Interestingly, we found 97 individuals (47%) with some form of thalassemia minor, 39 with α-thalassemia (18.9% of total population), 45 with β-globin mutation mainly Hb E (21.8%), and 13 with combined α and β globin abnormalities (6.3%). None of these individuals had genotypes found in thalassemia diseases such as Hb H disease or Hb E/β-thalassemia; therefore, they were classified as thalassemia minor and subsequently used for further analysis. Details of all comprehensive genotype data are shown in Table 1. Infants with thalassemia minor had no history of blood transfusion and no hepatosplenomegaly.

Table 1. Summary of globin genotypes found in 97 infants with thalassemia minor in this study.

We found no significant differences in all clinical characteristics: age, gender, growth and nutrition normal iron and those with IDA to see the effects of parameters, and iron markers such as SF, TS, and hepcidin levels. The exception was having a family history of anemia or thalassemia being more common in infants with thalassemia minor (43.3%) versus those without thalassemia (12.8%): Table 2. In our logistic regression analysis, having a history of anemia or thalassemia showed an increased risk of thalassemia minor in infants with an odds ratio of 5.18 (95% CI: 2.60-10.33), p = 0.001. Notably, the number of infants with both thalassemia minor and IDA at first diagnosis was higher than those with normal globin genotypes (32.0% vs. 20.2%); moreover, the number of infants with normal iron status and ID was significantly different among infants with and without thalassemia minor (p = 0.037): Table 2.

Table 2. Clinical and laboratory characteristics of all studied 206 infants with and without thalassemia minor.

The primary diagnosis of infants with IDA using Hb levels with SF and TS values was further confirmed when the majority responded to iron therapy. Thirty-four out of 53 IDA infants with (n = 31) and without thalassemia minor (n = 22) have received iron therapy, and all showed therapeutic response displayed as a significant increase across all red blood cell (RBC) parameters, compared to the baseline within their groups (p < 0.05). Of note, IDA infants without thalassemia minor (n = 18) had slightly greater increments in Hb and mean corpuscular volume (MCV) after iron therapy than IDA infants with some type of thalassemia minor (n = 16); however, there were no statistically significant differences between groups: Supplementary Table 1.
Iron status in infants with or without thalassemia minor. We performed subgrouping analysis within the groups of infants with and without thalassemia minor to determine the effects of normal iron status and IDA on hematological parameters (Table 3). There were significant differences in Hb, hematocrit (Hct), MCV, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red blood cell distribution width (RDW) within both groups suggesting iron played a significant role in determining Hb, Hct, and red blood cell indices. With the exception of increased RBC counts and RDW, the other RBC parameters were lower in those with IDA versus normal iron. Interestingly, infants with thalassemia minor who had IDA (n = 31) displayed statistically lower values in MCV, MCH, with the higher RDW: Table 3.

Table 3. Laboratory parameters of infants with and without thalassemia minor who had either normal iron status or iron deficiency anemia.

This suggests that the coinheritance of globin mutations can have epistatic hematological effects on top of any primary effects on iron status.
Effects of thalassemia minor on hematologic and iron parameters. We further compared those infants with thalassemia minor. Coinheritance of thalassemia had significant effects on Hb, Hct, RBC, MCV, MCH, RDW, but not MCHC, solely in those with normal iron status: Supplementary Table 2. However, we found only RBC and MCV to be significantly different in infants who already had IDA, suggesting this epistatic effect took place only within those two parameters. In addition, we found an effect of iron on significantly decreased levels of Hb A2 in infants without thalassemia minor but not in those with thalassemia. On the other hand, the basal Hb F in infants with thalassemia minor was generally higher than those without, suggesting a delay in globin switching, one of the consequences of globin abnormality.[43,44] Iron status does not appear to be significantly associated with the levels of persistent Hb F expression within the groups of both infants with and without thalassemia minor (Table 3).
Effects of thalassemia minor on hepcidin expression. We compared serum hepcidin, serum ferritin, and TS in infants without thalassemia having normal iron status to those having different types of thalassemia minor: Figure 1. Measurements for each group are in Supplementary Table 3A (iron replete) and Supplementary Table 3B (iron deplete). Levels of hepcidin appeared to be slightly lower in those with thalassemia minor and lowest in those with combined α and β globin mutations. This was consistent with the slightly higher serum ferritin and TS seen in those with combined thalassemia minors, although without statistically significant differences. No differences within these parameters were found between groups or by gender (data not shown).

Figure 1. Comparison of serum ferritin (A), transferrin saturation (B) and serum hepcidin (C) between normal infants (NL) and infants with 3 subgroups of thalassemia minor; α-thalassemia trait, β-thalassemia trait or hemoglobin (Hb) E trait, and combined α- and β-globin mutations.
#All infants shown here were determined to have normal iron status. The difference between each parameter of infant groups was analyzed using one-way analysis of variance (P). Note: NL: normal; a: α-thalassemia trait, b: β-thalassemia trait or hemoglobin (Hb) E trait, and c: combined α- and β-globin mutations. O: infants with thalassemia minor who had iron deficiency (ID); X: infants with thalassemia minor who had iron deficiency anemia (IDA).

We found the prevalence of thalassemia minor (carrier) in nearly half of the infants, at 47%. It was higher than previous reports of 30-40%.[13,15] One of the main reasons was the DNA testing used in our current study was far more comprehensive than the approaches such as cord blood hemoglobin studies, hemoglobin typing, etc. used 20 years ago. This is consistent with a report on thalassemia prevalence by Viprakasit V et al. in 2009,[45] stating that Hb E trait was the most common type of thalassemia minor[14,15,45] in Thailand, with a frequency up to 50-60% in Southeast Asia.[14] We found no individuals with thalassemia disease. This may point to the effectiveness of Thailand’s prevention and control program that screens for thalassemia carriers in pregnant women and their partners in order to identify the genetic risk of severe thalassemia syndromes.[16] Therefore, our studied population is likely to represent relatively “healthy” infants receiving routine health care and are a primary target for the iron supplementation program.
A previous study of β-thalassemia traits noted the presence of mildly increased erythropoiesis, as seen through elevated erythropoietin levels.[46] It has also been observed that adults with α- or β-thalassemia traits have shown increases in soluble transferrin receptors or erythropoietin concentrations, indicating ineffective erythropoiesis and increased erythropoietic drive leading to hepcidin suppression and upregulated iron absorption. Prior studies in India and Iran examining the iron status of adults with β-thalassemia traits concluded that β-thalassemia traits had higher serum ferritin than the controls, representing an advantage in iron balance.[47,48] These particular findings did not concur with others, which had stated that ID might commonly coexist with thalassemia traits.[20,49,50] These conflicting results have caused uncertainty in iron supplementation strategies for areas with a high prevalence of hemoglobinopathy. Of note, a universal iron supplementation program for infants might not be directly applicable to countries where differences in the genetic background of thalassemia, especially Hb E and α-thalassemia traits are uncommon and β-thalassemia traits are more frequent.[51,52] With a potential increase in the risk of iron overload for individuals with thalassemia minors, universal iron supplementation programs remain a point of contention.
A recent community study of 1821 Sri Lankan schoolchildren aged 8-18 years (48.3% males) from the Oxford group has shown that this might be the case for those with β-thalassemia traits.[28] Eighty-two β-thalassemia carriers with iron-replete had evidence of increased erythropoiesis, a slight but significant reduction in hepcidin, and suppression of hepcidin out of proportion to their iron stores: lower hepcidin-ferritin ratio compared with non-carrier controls (n = 176 with normal MCV and MCH). Another Sri Lankan cross-sectional study of 2273 children (aged 12–19 years) from a total of 7526 students, reported the same effect in iron-replete α‐thalassemia carriers as compared to the non‐iron deficient controls without thalassemia minor (4.8 ng/mL vs 5.3 ng/mL, p = 0.02).[53] However, this was not observed in those with Hb E traits from both cohorts.[28,53] Based on these results, it has been proposed that a hepcidin cutoff of < 3.2 ng/mL could be used to select cases for iron supplementation in countries with high rates of thalassemia carriers.[53] Both studies were conducted in primary and secondary school students as this is the age group at which iron supplementation is given in Sri Lanka. However, the effects of being a thalassemia carrier on hepcidin suppression, as well as the risk of iron accumulation in younger cases with thalassemia minor, remain unclear.
Our study determined this iron supplement issue in infants with thalassemia minor. While we could not find significant hepcidin suppression in our infants with thalassemia minors as compared to previous studies, our results were somewhat in line with such findings. Most of our thalassemia minors were Hb E traits, and this condition did not show a significant enough globin imbalance leading to ineffective erythropoiesis and subsequent hepcidin suppression. Moreover, even for individuals with homozygous Hb E, we found no evidence of this effect. Our infants with α-thalassemia carriers also demonstrated no effects of hepcidin suppression, differing from the previous study.[53] This may be because our population was younger with remaining Hb F expression (Table 1 and 3) and had less globin imbalance and ineffective erythropoiesis per se. It is, therefore, possible the erythropoietic drive that suppresses hepcidin was not fully operative yet.
In addition, the normal physiology of hepcidin expression, especially within the first year of life, might be more dynamic. A recent study in late preterm infants (32-36 weeks gestation) described a physiologic decrease of hepcidin levels during the first four months of life to increase iron availability.[54] Another longitudinal study that followed 140 Spanish healthy and full-term infants found hepcidin levels increased from six to 12 months of age with hepcidin levels positively correlated with iron status.[55] These results suggested that, in normal babies, a regulation of hepcidin production is under development during the first year of life; this may also be true for infants with thalassemia. Therefore, the effects of ineffective erythropoiesis on hepcidin suppression in thalassemia traits are likely not fully apparent during the first year of their life. This warrants further study to define at what age this effect would first be identified.
We still found our infants with thalassemia minor having a high proportion of iron depletion (57.7%), similar to infants without thalassemia (61.5%); the number of infants with both thalassemia and IDA was even significantly higher than infants without thalassemia minor (32 vs 20.2%) (Table 2). Comparing with the two previous studies at well baby and well child clinics of different university hospitals, we reported markedly higher prevalence of IDA (25.7% VS 19% by Linpisarn et al and 14.3% by Tantracheewathorn et al).[56,57] Nevertheless, our prevalence was in disagreement with the Fifth National Nutritional Survey of Thailand in 2003 by Department of Health, Ministry of Public Health of Thailand which reported prevalence of anemia in 6–11-month-old infants up to 56.3%.[58] These discordances might be due to differences in geographic settings, methodology and socioeconomic level of the populations. Since Thailand is an endemic area of thalassemia and malaria, we urge exploration and explanation of all causes of anemia, including inherited and acquired, and the application of proper management. Additionally, the long-term irreversible neurodevelopmental consequences of ID,[59,60] as well as the extremely critical diagnosis and prevention of ID and IDA in infants, are well-known and published, including preventive strategies of iron deficiency anemia and other micronutrient deficiencies in reproductive-aged and pregnant women which might be affecting their child health and developmental outcomes.[61] Universal screening for anemia by Hb concentrations at approximately 12 months of age, as recommended by the American Academy of Pediatrics (AAP)[62] and the Royal College of Pediatricians of Thailand & Pediatric Society of Thailand, might not be suitable for Thailand. We would, instead, strongly recommend screening and early detection of ID and anemia in infants aged 6-12 months in well-childcare settings by CBC, RBC morphology and the proper iron parameters. Thus, the coexisting causes (such as chronic hemolysis and inflammation) and possible risk factors of ID need to be further identified, especially unresponsiveness to oral iron therapy, including inadequate iron in complementary feeding (maternal preference for staple foods), micronutrient deficiency, intestinal infection, parasitic infestation, and malabsorption.[63] Nevertheless, infants with thalassemia minor who have IDA or ID would benefit from proper iron supplementation.
Interestingly, infants with coexisting thalassemia minor and IDA had significantly reduced Hb, MCV, MCH, and MCHC with increased RDW versus those having thalassemia minor with normal iron (Table 3). These findings were consistent with previous studies in India where MCV and MCH were significantly lower in adults with combined thalassemia traits and IDA than with either of these conditions.[49] Moreover, discordance between RBC count and RDW was also observed in infants with coexisting thalassemia minor and IDA as described in recent studies.[64,65] In addition, diminished HbA2 levels in patients with concomitant β-thalassemia traits and iron deficiency have been observed.[51,66] We believe our RBC indices to present a comprehensive analysis of thalassemia carriers in this age group. Our findings could be useful as references.
Among 36 thalassemia minor infants with anemia, we found five cases who did not have coexisting IDA, including infants with two α-thalassemia traits (-α^3.7/αα and --^SEA/αα), one β-thalassemia trait, one Hb E trait and one homozygous Hb E. This suggested that α- and β-thalassemia traits may be the cause of mild anemia in some infants. Accordingly, anemic infants unresponsive to oral iron therapy should be investigated for thalassemia rather than continuously undergoing long-term iron therapy by default, as toxicity or other side effects may develop. A familial history of anemia or thalassemia, as shown herein, was found to be strongly associated with thalassemia minor in offspring and could be used to diagnose future cases early.

Justification of Universal Iron Supplementation for Infants 6-12 months in Regions with a High Prevalence of Thalassemia