The Effect of Co‐Existing Iron Deficiency on HbA2 Level and the Diagnosis of Beta Thalassemia Trait

Iron deficiency has been found to affect hemoglobin A2 (HbA2) values in HPLC. This can be an issue for thalassemia screening laboratories where there is heavy reliance on increased Hb A2 levels for diagnosis of heterozygous thalassemia state. In resource constrained countries like India this could be real challenging where iron deficiency is widespread and facilities for molecular confirmation in borderline HbA2 values is generally unavailable. It was a prospective study done in a tertiary care center over 18 months. All consecutive patients (n = 164) presenting with microcytic hypochromic anemia on peripheral smear were included for further investigations out of which 92 were found to have pure iron deficiency (Hb < 12 Gm/dL with ferritin less than12 ng/ml) on iron parameters. These patients were divided into two groups, Group A with Hb < 9 g/dl and Group B with Hb > 9 g/dl. Common hematological parameters, iron indices and HbA2 levels were analysed in these two group of patients at baseline and after 3 months of documented oral iron therapy. Chi-square and Pearson tests were used for statistical analysis and a P- value of < 0.05 was considered statistically significant. As expected iron deficiency was found more prevalent in females (72%) than in males(28%). Mean pre -treatment and post - treatment hemoglobin of patients in group A was 8±0.5 gm/dl and 11.3±1.1gm/dl respectively and in group B was 10.2±0.6 g/dl and 11.5±1 g/dl showing positive correlation. Mean pre treatment and post treatment HbA2 levels of patients in group A were 1.8±0.5% and 2.4±0.5% respectively showing statistically significant change after iron therapy (P< 0.0001) but mean pre treatment and post treatment HbA2 levels of patients in group B were 2.1±0.4% and 2.2±0.5% respectively . this change post therapy was statistically insignificant(P=0.1517). The change in HbA2 levels was statistically insignificant for patients with mild / moderate iron deficiency anemia (Hb > 9 Gm/DL). Thus diagnosis of β thalassemia trait will not be difficult in patients with concomitant mild iron deficiency anemia but patients with severe iron deficiency anemia should first be treated with iron supplements for correct diagnosis of β Thalassemia trait especially patients with borderline Hb A2 levels.

Background: Thalassemia is an inherited hemoglobin disorders in which there is reduction or absence of α or β chains due to mutations in α or β genes. Microcytic hypochromic anemia is hallmark of beta thalassemia trait and the patient is usually asymptomatic. It is important to differentiate it from iron deficiency anemia (IDA) which also shows similar peripheral blood picture. The diagnosis is based on quantification of HbA2 and serum ferritin. If the level of HbA2 is more than 3. 5%, then diagnosis of beta thalassemia trait is made. The problem arises with borderline HbA2 i. e 3.1% - 3.4%. There is also diagnostic difficulty when concomitant iron deficiency anemia is present.
 Objectives: To determine the diagnostic accuracy of RDWI in borderline HbA2 to distinguish the beta thalassemia trait from IDA and to diagnose beta thalassemia trait when genetic analysis is not easily available.
 Methods: It was a cross sectional validation study. A total of 90 patients were included in this study, having HbA2 between 3.1-3.4%. Tests for serum ferritin, serum iron and total iron binding capacity (TIBC) were done on all samples. RBC morphology, RBC count, mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC) and RDW of all samples were noted. RDWI of all samples was calculated. Mutational analysis by ARMS- PCR was done for confirmation of β thalassemia trait.
 Results: Out of 90 samples with borderline HbA2, 30 cases were diagnosed as β thalassemia trait. Out of these, 15(50 %) had concomitant IDA along with beta thalassemia trait and 15 were non iron deficient beta thalassemia carriers. 43 samples were not iron deficient and were also negative for β thalassemia trait, whereas 17 samples had only IDA without beta thalassemia trait. The sensitivity of RDWI was 76.6% and specificity was 56.6% in borderline HbA2.
 Conclusion: RDWI is a good indicator to differentiate between IDA and beta thalassemia trait (sensitivity 76.6% specificity 56.6%). But in all cases of borderline HbA2 genetic analysis should be done to see the mutation and to confirm the diagnosis.

Variability In Hb A2 levels among Individuals with Beta-Thalassemia Trait: Is Iron Deficiency Associated with Abnormally Low Hb A2?

Borderline hemoglobin A2 levels in northern Thai population: HBB genotypes and effects of coinherited alpha-thalassemia

Background: Thalassemia trait screening often relies on the quantification of Hemoglobin A2 (HbA2) levels, which are typically elevated in beta-thalassemia carriers. However, iron deficiency, a highly prevalent condition in India, especially among females, has been postulated to reduce HbA2 levels, potentially leading to falsenegative screening results. Understanding the impact of iron deficiency on HbA2 values is critical to ensuring accurate detection of thalassemia carriers, especially in population-level screening programs. Objectives: To evaluate the effect of iron deficiency on HbA2 levels and determine whether iron deficiency can mask the diagnosis of beta-thalassemia trait in individuals undergoing screening. Methods: This observational cross-sectional study was conducted in the Department of Pathology, Darbhanga Medical College and Hospital, Bihar, from August 2022 to July 2023. A total of 130 individuals aged 18–40 years undergoing anemia evaluation or thalassemia screening were included. Participants were grouped into irondeficient and non-iron-deficient groups based on serum ferritin, transferrin saturation, and complete blood count findings. HbA2 quantification was done using high-performance liquid chromatography (HPLC). Statistical analysis was performed to assess the correlation between iron status and HbA2 levels. Results: Out of 130 participants, 72 were iron-deficient while 58 had normal iron stores. The mean HbA2 level in the iron-deficient group was 2.3 ± 0.4%, significantly lower than the non-iron-deficient group which had a mean HbA2 level of 3.1 ± 0.5% (p < 0.001). Among iron-deficient individuals with suspected thalassemia trait, HbA2 levels were found to be within normal or borderline ranges, indicating the possibility of masked diagnosis. Upon iron repletion and re-evaluation in a subset, HbA2 levels rose significantly, unmasking latent betathalassemia trait in some cases. Conclusion: Iron deficiency significantly lowers HbA2 levels and may obscure the diagnosis of beta-thalassemia trait in screening programs. Screening for thalassemia should either follow correction of iron deficiency or incorporate parallel iron status assessment to avoid misclassification. This is particularly relevant in highprevalence regions like Bihar where iron deficiency anemia is common.

Background. Coexistence of iron deficiency anemia (IDA) and beta thalassemia trait (BTT) has been the topic of few studies. However, no study from our country was found evaluating the effect of iron therapy in patients with concomitant IDA and BTT. Methods. Over a period of two years, 30 patients with concomitant IDA and BTT were included. All the patients had a complete blood count, serum iron studies, and thalassemia screening using BIORADTM hemoglobin testing system. The patients received oral iron therapy in appropriate dosages for a period of twenty weeks, after which all the investigations were repeated. Appropriate statistical methods were applied for comparison of pre- and posttherapy data. Results. All except two patients were adults with a marked female preponderance. Oral iron therapy led to statistically significant improvement in hemoglobin, red cell indices (P < 0.05), and marked change in serum iron, ferritin, and HbA2 levels (P < 0.001). There was a significant reduction in the total iron binding capacity levels. Conclusion. The present study shows the frequent occurrence of iron deficiency anemia in patients with beta thalassemia trait, which can potentially confound the diagnosis of the latter. Hence, iron deficiency should be identified and rectified in patients with suspicion of beta thalassemia trait.

Objectives: We aimed to evaluate the effect of low serum ferritin levels on HbA2 values in BTT patients. Study Design: Cross-sectional study. Setting: Pathology department of University Medical &amp; Dental College Faisalabad. Period: August, 2018 to July, 2019. Materials &amp; Methods: One hundred and thirty seven subjects were included in the study after written informed consent. Those with serum ferritin &lt; 10µg/L were taken as iron deficient. Based on serum ferritin levels, we divided our study participants into two groups (Group A Vs Group B). As ferritin is considered an acute-phase protein, 25/137 participants with leukocytosis were excluded from statistical analysis. We measured serum Ferritin on Cobas 6000 e611 and we assessed the red cell parameters on Sysmex (seven part differential XN 1000). Hb variants were analysed through High performance liquid chromatography (HPLC) based technique of BioRad D10. Results: After excluding 25 subjects with high Total leukocyte count (TLC), we are left with 112 subjects. We observed 26 participants in group A with Iron deficiency and 38 in group B with no Iron deficiency. Mean±SD serum ferritin in iron deficient group was 7.25±1.95 as compared to non-iron deficient group (87.63±7.35). Mean HbA2 value in group A was 4.56±0.04 and in group B it was 5.80±1.06 with significant statistical difference of P=0.0188. We also observed significant difference in the mean values of other Red cell indices (MCV, HCT MCHC, MCH) except for RBC count and RDW. Conclusion: This study shows that ID may reduce HbA2 levels. Overall, it does not essentially preclude the identification of BTT. It is recommended that Iron deficiency should be considered before measuring HbA2 levels in BTT.

Elevated Hb A2 level (≥4.0%) is considered to be reliable parameter to identify β-thalassemia (β-thal) carriers. However, some β-thal carriers have been misdiagnosed as their Hb A2 levels are below 4.0%. In addition, coinheritance of α-thalassemia (α-thal) and β-thal might affect Hb A2 levels. Therefore, the aim of this study was to investigate the mutations of β- and α-globin genes in individuals with borderline Hb A2 levels in Thailand. Three hundred samples from individuals with Hb A2 levels of 3.5–3.9% were collected for molecular diagnosis of β-globin gene mutations. In addition, the α0-thal, α+-thal, Hb Constant Spring (Hb CS, HBA2: c.427T>C), and Hb Paksé (HBA2: c.429A>T) diagnostics were also performed. Sixteen samples (5.33%) had β-globin gene mutations, and codon 41/42 (–TTCT) (HBB: c.126_129delCTTT) was the most prevalent mutation. Ninety-eight samples (32.67%) had α-globin gene mutations including four Hb H (β4)-Hb CS disease, two Hb H disease, 13 heterozygous α0-thal, 11 homozygous α+-thal, two α+-thal/Hb CS, one α+-thal/Hb Paksé, 61 heterozygous α+-thal, and four Hb CS. Furthermore, seven cases of β-thal carriers coinheriting α-thal were observed, and five of them carried Hb H disease. High prevalence of both α- and β-thal in subjects with borderline Hb A2 levels suggested that molecular diagnosis of α- and β-thal should be performed, especially in a high prevalence area of thalasssemia carriers, for accurate diagnosis and genetic counseling to prevent and control new severe thalassemia cases. Moreover, β-thal carriers who coinherited Hb H disease might have reduced Hb A2 levels, leading to a misdiagnosis of β-thal in analysis programs.

remaining 161 cases with an HbA2 level 3.5% constituted the non-BTT group. In the BTT group 3 cases had concomitant iron deficiency. In the non-BTT group 120 cases had a serum ferritin level <15 ng/mL and were diagnosed as IDA; of the remaining 41cases, 12 had associated chronic illness indicative of ACD (anemia of chronic disease) and in the other 29 the cause of microcytosis could not be identified.

β-Thalassemia is an autosomal recessive single gene disorder with mutations in the b-globin gene, resulting in faulty b-chain production, insufficient erythropoiesis, abnormal destruction of red blood cells, and consequent anemia.[1] Patients with thalassemia major are diagnosed in the early years of life and require lifelong blood transfusions and might also need subsequent iron chelation therapy. Patients with thalassemia intermedia (TI) can present with moderate anemia that does not require blood transfusions.[2] Dry eye has been reported to be one of the ocular associations of thalassemia, along with cataract, color blindness, visual field defects, pseudoxanthoma elasticum, and retinal pigment epithelium degeneration.[1,3,4] Case Report A 2-year-old boy was brought by his mother with complaints of intolerance to light. Ocular examination showed diffuse corneal punctate epithelial erosions, immediate break up of tear film, corneal haze, and severe intolerance to light. The systemic evaluation revealed frontal bossing and malar prominence [Fig. 1c] suggestive of hemolytic facies, pallor, cutaneous xerosis [Fig. 1d], hepatosplenomegaly, developmental delay, and microcephaly. Thalassemia screening performed at 1 year of age showed microcytic hypochromic anemia, anisopoikilocytosis, and tear-drop cells on a peripheral blood smear. Although his serum ferritin level was 20.2 ng/ml, his Mentzer index (11.2) was strongly suggestive of thalassemia. High-performance liquid chromatography (HPLC) showed a low fetal hemoglobin (<0.8%) and elevated HbA2 level (3.4%). Following this evaluation, the child had been started on oral iron and folate therapy. His mother also had chronic dry eye and typical facial features of thalassemia [Fig. 1e] along with a history of chronic anemia for which she was put under treatment with oral iron supplements by her treating physician. Her ocular examination revealed pallor of the palpebral conjunctiva, decreased tear film height, diffuse corneal epithelial erosions with scarring, and peripheral conjunctivalization [Fig. 1a and b]. Fundus examination was normal in both eyes, and the best corrected visual acuity was 6/12 in the right eye and 6/18 in the left eye. Both the mother and child were treated with preservative-free eye drops and lubricants along with supportive lifestyle measures. In concurrence with the clinical and hematological picture, a differential diagnosis of TI was made, and they were referred to a higher center for further management.Figure 1: (a and b) Diffuse slit-lamp image showing a lusterless cornea, corneal scarring, and peripheral conjunctivalization. (c) Image of 2-year-old showing frontal bossing. (d) Dry and scaly skin (cutaneous xerosis) of lower limbs. (e) Image of the mother showing frontal bossing and malar prominenceDiscussion Features of the thalassemia trait and TI can closely resemble iron deficiency anemia (IDA). However, differentiating the two conditions is important as hemoglobin may not improve with oral iron therapy in TI and might in turn precipitate an iron overload. Major differentiating points between IDA and TI are that TI shows normal serum ferritin levels and raised HbA2 levels (>3.2%), and anemia is unresponsive to iron therapy.[2] Studies have also shown that hepcidin, which is involved in the regulation of iron absorption and transport is suppressed in TI, leading to increased intestinal iron absorption, which can be worsened by prolonged oral iron therapy.[5] Conjunctival squamous metaplasia, goblet cell loss, and lower tear film break-up time have been reported in thalassemia patients with ocular surface disorders.[6] It has been postulated that accumulation of iron in the lacrimal glands has cytotoxic effects, consequently leading to aqueous deficiency and dry eye.[3] With advancing diagnostic and treatment modalities for thalassemia, it is imperative to identify associated ocular abnormalities to ensure comprehensive management. Differentiation between IDA and TI is crucial to avoid mismanagement of the latter with oral iron therapy as an iron overload might possibly precipitate ocular surface disorders. Conclusion Appropriate and timely management of dry eyes is necessary to improve patient comfort and alleviate the sequelae of ocular surface disorders. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Aravind eye hospital, Pondicherry. Conflicts of interest There are no conflicts of interest.

The most common differential diagnoses of microcytic hypochromic anaemia are iron deficiency anaemia and beta thalassemia. Globally, thalassemia affects approximately 4.4 out of every 10,000 live births whereas iron deficiency anaemia comprises half of all anaemia worldwide as per world health organization. The definitive diagnosis of beta thalassemia trait and iron deficiency anaemia requires haemoglobin analysis and iron studies respectively, which are not possible to perform in all suspected cases especially in resource limited settings. The study aims to evaluate the reliability of mentzer index in differentiating beta thalassemia trait from iron deficiency anaemia. This was a cross sectional, observational study done on 59 patients each of beta thalassemia trait and iron deficiency anaemia from August 2019 to July 2020. Patients who were found to be having iron deficiency anaemia diagnosed by iron studies and beta thalassemia trait diagnosed by Hb electrophoresis were enrolled in the study using simple random sampling technique. Mentzer index correctly identified 95.76% of overall patients. Area under receiver operating characteristic curve was 0.993 (95% CI, 0.985-1.002, p<0.001). For beta thalassemia trait, mentzer index showed a sensitivity of 93.2%, specificity of 98.3%, positive predictive value of 98.2%, negative predictive value of 93.5%; while for iron deficiency anaemia, sensitivity of 98.3%, specificity of 93.2%, positive predictive value of 93.5% and negative predictive value of 98.2%. Youden's index was 91.5. The findings of the present study make mentzer index a reliable screening method, especially in a resource poor setting, like Nepal. Further confirmation by gold standard tests is recommended.

BackgroundThe diagnostic criterion for beta thalassemia trait (BTT) is elevated Hb-A2 levels. Iron deficiency anemia (IDA) reduces the synthesis of Hb-A2, resulting in reduced Hb-A2 levels, so patients with co-pathological conditions BTT with IDA, may have a normal level of Hb-A2. Many socio-economic factors like unawareness, poor diagnostic facilities, and cost of molecular diagnosis (for screening purposes) result in interpretation of these subjects as normal.MethodsVenous blood samples from 200 unmarried females having a family history of thalassemia were collected, and basic hematological parameters, hemoglobin electrophoresis, and molecular analysis for beta thalassemia were done. Patients with IDA and patients with co-pathological conditions BTT and IDA were treated with oral iron. These subjects were then followed for a period of 20 weeks.ResultsOf the 200 females, 34 were found to be anemic. Hemoglobin electrophoresis identified 16 of these patients as BTT. Molecular analysis of all patients confirmed this diagnosis, but identified 8 additional patients with BTT. Eight patients that were not detected with hemoglobin electrophoresis were found to have co-pathology of BTT with IDA.ConclusionPatients with the co-pathological condition BTT with IDA may be interpreted as being normal, as they have normal Hb-A2 levels. These misdiagnosed subjects when marry with BTT have the potential to produce beta thalassemia major in offspring. This is one of the factors playing a major role in the propagation of beta thalassemia gene in Pakistani population, and become a serious hindrance for the thalassemia prevention program in Pakistan.

Beta thalassaemia is one of the most common inherited haemoglobinopathies, characterised by reduced or absent production of the beta globin chain. In India, the carrier frequency of thalassaemia is estimated to be 3-4%. The prevention of Beta thalassaemia is the best strategy, and this can be achieved through carrier screening and prenatal diagnosis. Carriers of beta thalassaemia can be easily identified using haematological parameters such as complete blood count and High Performance Liquid Chromatography (HPLC) for haemoglobin analysis. The characteristic findings observed in thalassaemia carriers include microcytosis, hypochromia, with a Mean Corpuscular Volume (MCV) of less than 80 fL and Mean Corpuscular Haemoglobin (MCH) of less than 28 pg. They also present with elevated levels of HBA2 (α2δ2) ≥3.5%. Carrier screening for beta thalassaemia primarily relies on the observation of elevated HbA2 levels. However, in rare cases, some carriers can have normal HbA2 levels, leading to false-negative screening results. In a case involving a married couple who underwent routine preconceptional screening by complete blood count and HPLC for thalassaemia screening, the male partner had elevated HbA2 levels (5.2%), while the female partner had normal HbA2 levels (1.6%). Molecular testing revealed that the male partner was heterozygous for the Intervening Sequence (IVS) 1-5 (G&gt;C) mutation, while the female partner was found to be heterozygous for the CD41-42 (-CTTT) mutation. It is important to consider molecular testing of the HBB gene in couples, even if one partner is a carrier and the other partner has normal or borderline HbA2 levels.

Borderline HbA2 levels: Dilemma in diagnosis of beta-thalassemia carriers

KLF1 gene mutations cause borderline HbA2