Is HbA2level a reliable diagnostic measurement for β‐thalassemia trait in people with iron deficiency?

Microcytic anemia is commonly caused by either iron deficiency, β-thalassemia trait (BTT) or α-thalassemia trait. Elevated HbA2 level (≥3.5%) is a well-established screening test for BTT 1. Conflicting reports have led to confusion about the reliability of this test to screen for BTT in the presence of iron deficiency. In this study, 444 people with BTT were confirmed by DNA-based genotyping. HbA2 levels were assessed by high performance liquid chromatography (HPLC). Individuals were classified as “iron-deficient” or “non-iron-deficient” based on serum ferritin level. The mean HbA2 (5.3%) in individuals with serum ferritin <15 μg/L was lower than those who are not iron-deficient (5.6%; P = 0.004). Nevertheless, HbA2 in individuals with serum ferritin <15 μg/L ranged from 4.2 to 6.2%, with none <3.5%. Multiple linear-regression analysis revealed a significant association of lower HbA2 with β+-thalassemia mutation and serum ferritin <15 μg/L. Thus, in this large cohort of individuals with BTT, serum ferritin <15 μg/L was associated with a small decrease in HbA2. Nonetheless, individuals with overt iron deficiency and BTT consistently had elevated HbA2 (≥3.5%) indicating that HbA2 remains a reliable test for BTT screening in the presence of iron deficiency. Beta-thalassemia is of public health importance in many parts of the world. Screening for β-thalassemia trait (BTT) is necessary for family counseling. The potential causes of “false-negative” testing must be understood (i.e., circumstances in which the individual being tested has BTT but HbA2 is not elevated). One example is (δβ)0-thalassemia in which there is decreased production of δ-globin chains as a result of δ-globin gene deletion 2. Carriers of β+-thalassemia mutations reportedly have lower HbA2 than carriers of β0-thalassemia mutations. There is a small subset of individuals with BTT and normal δ-globin genes who have normal HbA2 levels 3, 4. Some reports have suggested a lower than expected HbA2 in individuals with BTT who are iron-deficient 5-9. Other studies have found no significant effect of iron deficiency on the level of HbA2 in BTT 10, 11. These divergent reports have led to confusion about the reliability of this test in the presence of iron deficiency. In this study, we evaluated more than 400 Chinese individuals who were recruited for a genome-wide association study of β-thalassemia in Hong Kong 12, 13. Subjects were documented to have BTT by molecular genotyping. Serum ferritin measurements and hemoglobin analysis by HPLC were done, thus allowing us to analyze the possible relationship between low serum ferritin and HbA2 level. Furthermore, we sought to evaluate other clinical and laboratory determinants that may be related to HbA2 variability in this population. Of the 444 individuals with BTT, 252 (57%) were female. The mean age was 42 years, ranging from 1- to 78-years-old. The four most common β-thalassemia mutations were: Codons 41/42 (194 individuals); IVSII-654 (112); Nt – 28 (44); Codon 17 (41). HbA2 ranged from 2.4 to 8.1%. Six individuals had a HbA2 level below 3.5% (range 2.4–3.1%). Of these, five had the Chinese (Aγδβ)0-thalassemia deletion. As heterozygous deletion of the δ-globin gene would be expected to normalize HbA2 levels, these subjects were excluded from subsequent analyses. The remaining 439 individuals had HbA2 levels ranging from 2.7 to 8.1% (5.6% ± 0.5%; Fig. 1). Mean HbA2 in females (5.5% ± 0.5%) was lower than that in males (5.7% ± 0.5%; P = 0.0002). Distribution of hemoglobin A2 values in the 439 individuals analyzed. Individuals with serum ferritin <15 μg/L in red, individuals with serum ferritin ≥15 μg/L in blue. The mean serum ferritin was 181 ± 195 μg/L (range 2–1,377 μg/L; Supporting Information Fig. 1), with lower levels for females (94 ± 119 μg/L) than for males (296 ± 216 μg/L; P < 0.0001). Serum ferritin met the criteria for iron deficiency (<15 μg/L) in 30 females and no males. If the threshold for diagnosis of iron deficiency was changed to serum ferritin <30 μg/L, 67 individuals met this criteria (65 female and 2 male). Thirty females with serum ferritin less than 15 μg/L had significantly lower hemoglobin concentration (10.3 ± 1.2 g/dL) and mean corpuscular volume (MCV) (64.9 ± 5.4 fL) when compared with individuals who had serum ferritin of 15 μg/L or higher(Hb 11.9 ± 1.1 g/dL; MCV 67.0 ± 4.9 fL; P < 0.0001 and P = 0.03, respectively). This provides supporting evidence of iron-deficient hematopoiesis in the low serum ferritin group. The HbA2 in those with serum ferritin less than 15 μg/L was 5.3% ± 0.5% (range, 4.2–6.2%) as shown on Fig. 1. For those with serum ferritin ≥15 μg/L, their HbA2 was 5.6 ± 0.5% (P =0.004). Sixty-seven individuals with serum ferritin below 30 μg/L had significantly lower mean hemoglobin concentration (10.8 ± 1.0 g/dL) when compared with those who had serum ferritin of 30 μg/L or higher (12.0 ± 1.1 g/dL; P < 0.0001). There was no significant difference between their MCV's (66.5 vs. 67.0 fL, respectively; P = 0.5). The HbA2 in those with serum ferritin less than 30 μg/L was 5.4% ± 0.4% (range, 4.1–6.4%). For those with serum ferritin ≥30 μg/L, their HbA2 was 5.6% ± 0.5% (P = 0.01). There was no significant difference in HbA2 values in individuals heterozygous for the single rightward (3.7) or leftward (4.2) α-globin gene deletion or deletion of both α-globin genes in cis of the Southeast Asian type when compared with individuals with four α-globin genes (data not shown). After excluding individuals with (Aγδβ)0-thalassemia genotype, there was a significant difference between the variances among the remaining individuals who are carriers of different β-thalassemia mutations, F(10, 424) = 9.11, P < 0.0001. Their HbA2 level and β-thalassemia genotypes are shown in Supporting Information Table 1. Multiple linear-regression analysis of association between low HbA2 and several variables revealed a significant association with low serum ferritin less than 15 μg/L (P = 0.02) and β+-thalassemia mutation (P < 0.001). There was no statistically significant association with gender, hemoglobin level, HbF level, or reticulocyte percentage. In this study of 444 individuals with BTT, 30 had overt iron deficiency (serum ferritin <15 μg/L), all of whom had HbA2 levels ≥3.5% as expected for BTT. Six individuals had HbA2 below 3.5%; all but one were carriers of (Aγδβ)0-thalassemia deletion. Serum ferritin less than 15 μg/L was accompanied by evidence of iron-deficient hematopoiesis, and was associated with a small but significant decrease in HbA2 (mean HbA2 of 5.3% vs. 5.6% in non iron-deficient individuals; P = 0.004). Although multiple linear regression analysis confirmed that ferritin below 15 μg/L was associated with lower HbA2 levels, β+-thalassemia mutation type was more highly associated with lower HbA2 values. The trend toward lower HbA2 values in β+-thalassemia was reported by Huisman in 1997 14. It is postulated that HbA2 is increased in β-thalassemia trait because of reduced β-globin chain synthesis, leading to an excess of α-globin chains and subsequent formation of HbA2 (α2δ2). It would follow that, if β-globin chain production was less impaired as in β+-thalassemia, there would be less excess of α-globin chains and therefore lower HbA2. In addition it is postulated that because β+-thalassemia usually involves a promoter mutation, a lack of competition for transcription factor and the β-LCR will lead to higher HbF and HbA2. Regarding the association of HbA2 and gender, an association of lower HbA2 levels in females was apparent using the student's t-test. This association was not apparent on multivariate analysis, suggesting that the association between gender and HbA2 in our study population was better explained by other factors (e.g., β-thalassemia mutation type and/or serum ferritin). Reports of studies on the relationship between iron deficiency and HbA2 levels in BTT are conflicting (Supporting Information Table 2). This is likely a result of patient heterogeneity, study design, definition of iron deficiency, HbA2 level, and BTT. Criteria for diagnosis of β-thalassemia trait: Almost all previous studies used surrogate markers to identify BTT, such as red blood cell indices or HbA2 level, which could potentially have mis-identified individuals as having BTT and/or confounded interpretation of their results. Methods used to determine HbA2 and iron deficiency: Some methods of HbA2 measurement are unreliable. There is also variability between laboratories. Similarly, there are many methods used for determining iron store status. Bone-marrow hemosiderin staining is considered the gold standard, but requires bone marrow examination which is an invasive procedure. Serum ferritin is a simple and reliable method of evaluating iron stores. Only the two most recent studies used low serum ferritin as a marker of iron deficiency 9, 10. No studies assessed bone-marrow hemosiderin. It has been shown that serum ferritin level shows a better association with bone-marrow hemosiderin level than serum iron, with or without transferrin saturation 15, 16. Variation in β-thalassemia mutation: β-thalassemia mutations differ among racial and ethnic groups. If mutation genotype was not determined and taken into consideration in the analysis of HbA2 levels, associations could have potentially been erroneously attributed to iron deficiency. HbA2 levels can be low or normal in δβ-thalassemia trait, or when BTT is coinherited with a δ-globin gene mutation. If testing is not done for δ-globin abnormalities in BTT individuals with low HbA2 and iron deficiency, lower HbA2 might be erroneously attributed to iron deficiency. To the best of our knowledge, this is the first study of HbA2 and iron deficiency in individuals with BTT of Chinese ancestry. Each of the prior studies addressing this question evaluated different ethnic groups. Unknown genetic factors related to ethnicity may play a role. Previous studies of BTT and iron deficiency have noted that reduction in the HbA2 level is most pronounced in patients with profound iron deficiency. It has been postulated that synthesis of α-globin chains is inhibited, resulting in a relative deficiency of α-chains with which δ-chains can form HbA2 tetramer 1. We can not rule out the possibility that iron deficiency in our patient group was less severe than in previously reported groups. Based on the results of this study, elevated HbA2 is a reliable marker of BTT, even in the presence of iron deficiency. In patients suspected to have BTT, but in whom HbA2 is found to be <3.5%, testing for δ- and other globin gene abnormalities should be pursued. Parents and siblings with BTT were identified through β-thalassemia major or intermedia patients who were cared for at the Thalassemia Clinic in one of five hospitals in Hong Kong 12. All subjects signed an informed consent. Identifying information was removed from data files prepared for analysis. The research project was approved by the Institutional Review Boards of the Boston University School of Medicine, and each of the five participating hospitals in Hong Kong. All blood samples were delivered within 1 day after phlebotomy to the Division of Hematology, Department of Pathology at the Queen Mary Hospital in Hong Kong, where blood counts by Advia 120 counter, serum ferritin measurements, hemoglobin analysis by BioRad Variant II HPLC and DNA-based globin genotyping were performed 12. We assessed the variability in HbA2 and identified individuals with HbA2 below 3.5%. Individuals were also classified as “iron-deficient” or “noniron-deficient” based on their serum ferritin levels. The data was analyzed independently using two definitions of iron deficiency: (1) a serum ferritin level less than 15 μg/L, as per the recommendation of the World Health Organization 17, which has a sensitivity of 59% and specificity of 99% 18, and (2) a serum ferritin level less than 30 μg/L, which had a sensitivity of 96% and specificity of 100% in patients with microcytic anemia 19. The association of HbA2 levels with gender and iron deficiency was evaluated using a two-sample student's t-test. β-thalassemia mutation type and HbA2 level was evaluated using one-way analysis of variance (ANOVA) and post-hoc analysis with the Tukey's multiple comparison test (significance set for P < 0.05). The relationship of HbA2 with gender, hemoglobin level, HbF percentage, reticulocyte percentage, β-thalassemia mutation type, and/or low serum ferritin was evaluated using single and multiple linear regression analyses. Beta-thalassemia mutations were categorized as β0- or β+-type 20. All statistical analyses were performed with Stata 11 and GraphPad Prism 5.03 software. The authors are indebted to all individuals who agreed to participate in this research project, without whom this work would not have been possible. The authors thank Dr. S.Y. Ha, Dr. C. Kong Li, Dr. A.C.W. Lee, Dr. R.C.H. Li, Dr. C. Keung Li, and Dr. H.L. Yuen for allowing them to study their patients and family members, Yvonne Chu and Amanda Mok for their untiring effort in subject recruitment, and Amy Chan and Stella Tsang for laboratory testing. The authors thank Dr. Marcello Pagano for advice related to statistical analysis of the data. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Madeleine Verhovsek* , Chi-Chiu So , Timothy O'shea§, Geoffrey T. Gibney* §, Edmond S.K. Ma§, Martin H. Steinberg*, David H.K. Chui*, * Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, Department of Medicine, McMaster University, Hamilton, Ontario, Canada, Department of Pathology, University of Hong Kong, Hong Kong, § Department of Medicine, Yale University, New Haven, Connecticut.