Introduction

Transfusion-Dependent Thalassemia (TDT) is a hereditary hemoglobinopathy characterized by defective beta-globin chain synthesis, leading to severe anemia and necessitating lifelong blood transfusions1,2. While regular transfusions improve survival, they result in iron overload, a major complication that contributes to multi-organ dysfunction, including endocrine disorders such as hypothyroidism3,4. Iron overload is the major cause of morbidity, mortality and organ injury. Iron overload can cause iron to deposit and damage various organs like liver, heart, skin and causing thyroid dysfunction5,6. Iron deposition in endocrine glands, particularly the thyroid, disrupts hormone production, leading to subclinical or overt hypothyroidism, which further exacerbates morbidity in thalassemic patients7.

Iron chelation therapy (ICT) is essential to mitigate iron overload and its complications8. The three primary chelators—deferoxamine (DFO), deferiprone (DFP), and deferasirox (DFX)—vary in efficacy, administration routes, and side-effect profiles9,10. Despite their benefits, the impact of different chelators on thyroid function remains understudied, particularly in regions like Pakistan, where TDT prevalence is high11. Previous studies report hypothyroidism in 6–30% of TDT patients, influenced by factors such as chelation adherence, transfusion frequency, and serum ferritin levels4,12. However, comparative data on how specific chelators affect thyroid function are limited.

This study aimed to evaluate the effects of ICT on thyroid function tests (TFTs) in TDT patients in Mardan, Pakistan, and compare outcomes across different chelation regimens. By analyzing free triiodothyronine (fT3), free thyroxine (fT4), thyroid-stimulating hormone (TSH), and ferritin levels, we assessed the association between iron overload, chelation therapy, and thyroid dysfunction. Our findings contribute to optimizing chelation strategies to reduce endocrine complications in TDT patients, particularly in resource-limited settings where treatment adherence and monitoring remain challenging. While a longitudinal design is ideal for tracking temporal changes, this cross-sectional study was chosen to provide a timely, real-world comparison of thyroid function across the different iron chelation regimens currently in use within our clinical setting. The aim was to identify significant associations that could inform clinical practice and prioritize future interventional research.

Methodology

The research aimed to investigate the effects of iron chelation therapy (ICT) on thyroid function tests in beta-thalassemia major (BTM) patients. Samples were collected over a period of four months from Mardan Medical Complex, ensuring adherence to ethical guidelines and informed consent protocols.

Ethical consideration

Ethical approval was obtained prior to the commencement of the study to ensure compliance with institutional and national guidelines and was approved by the Research Ethical Committee of the Iqra National University, Peshawar, Pakistan, (Ref: INU/AHS/313-23). All participants, or their guardians in the case of minors, provided written informed consent after being thoroughly briefed about the study’s objectives, procedures, and potential risks. Confidentiality of participant data was maintained throughout the study, and all procedures were conducted in accordance with the Declaration of Helsinki.

Sample size

A total of 200 individuals were included in the study, divided into two groups: 100 beta-thalassemia major patients who had been receiving iron chelation therapy for at least six months, and 100 beta-thalassemia major patients who had not received ICT. The sample size was determined to ensure sufficient statistical power to detect significant differences in thyroid function tests between the two groups.

Questionnaire

A structured questionnaire was designed to collect sociodemographic and clinical data from the participants. Information such as age, gender, medical history, duration of thalassemia diagnosis, frequency of blood transfusions, and type of iron chelator used was recorded. The questionnaire also included details about any comorbidities or medications that could influence thyroid function. This data was essential for correlating clinical parameters with laboratory findings.

Inclusion and exclusion criteria

Participants in the patient group were diagnosed with beta-thalassemia major and had been receiving regular blood transfusions and ICT for a minimum of six months. Exclusion criteria included individuals with chronic inflammatory conditions, other hematological disorders, or any acute illness that could affect thyroid function. The control group comprised beta-thalassemia major patients who have not received iron chelation therapy.

Blood sample collection

Venous blood samples (5 mL) were collected from each participant under aseptic conditions. The cubital fossa was cleaned with an alcohol swab, and blood was drawn using a sterile needle and syringe. The samples were divided into two tubes: an EDTA tube for complete blood count (CBC) analysis and a serum separator tube (SST) for biochemical assays. After collection, the samples were transported to the laboratory under controlled conditions to ensure stability.

Complete blood count (CBC)

The EDTA blood samples were analyzed using the CELL-DYN Ruby Hematology Analyzer, an automated system capable of measuring multiple hematological parameters. The CBC included measurements of red blood cells (RBCs), white blood cells (WBCs), hemoglobin levels, hematocrit, and platelet counts. The analyzer employs flow cytometry to provide accurate and reproducible results, which are critical for assessing the hematological status of thalassemia patients.

Sample preparation for CBC

Prior to analysis, EDTA blood samples were thoroughly mixed by gentle inversion to prevent clotting and ensure homogeneity. Samples stored at refrigerated temperatures were brought to room temperature before processing. The analyzer aspirated 150–250 µL of blood, depending on the mode of operation, and generated comprehensive reports within minutes. Quality control measures were implemented to validate the accuracy of the results.

Biochemical parameters analysis

The SST samples were centrifuged at 3000 rpm for three minutes to separate serum, which was then aliquoted into Eppendorf tubes for further analysis. Biochemical assays included thyroid function tests (TSH, free T3, free T4), serum ferritin, liver function tests (ALT, ALP, GGT), and renal function tests (urea, creatinine). These tests were performed using the Architect i1000SR and ci4100 immunoassay analyzers, which employ chemiluminescence and enzymatic methods for high sensitivity and specificity.

Statistical analysis

Data were analyzed using SPSS version 25. Descriptive statistics were used to summarize demographic and clinical variables. The normality of data distribution was assessed using the Shapiro–Wilk test. Variables with a non-normal distribution (e.g., ferritin) were log-transformed prior to parametric testing, and key findings were validated with non-parametric tests. Paired t-tests compared thyroid function and ferritin levels between the patient and control groups. Pearson correlation assessed relationships between iron chelators and thyroid parameters, while one-way ANOVA evaluated differences among patients receiving different chelators (deferasirox, deferoxamine, deferiprone). A p-value < 0.05 was considered statistically significant.

Results

Table 1 summarizes the baseline characteristics of the study participants, comparing beta-thalassemia major (BTM) patients receiving iron chelation therapy (ICT) with BTM controls. Table 1 finding underscores the severe iron overload associated with chronic blood transfusions in thalassemia patients, reinforcing the necessity of ICT to mitigate iron-related complications.

Table 1 Baseline Characteristics of Study Participants.
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Table 2 presents the comparative analysis of thyroid function tests (TFTs) between beta-thalassemia major (BTM) patients receiving iron chelation therapy (ICT) and BTM controls. The results demonstrate significant differences in all measured thyroid parameters. Table 2 findings suggest that ICT may play a protective role in maintaining thyroid hormone homeostasis in BTM patients, potentially mitigating the hypothyroid effects of iron overload. The marked divergence in TFTs between groups underscores the clinical significance of endocrine monitoring in thalassemia management and highlights the potential benefits of chelation therapy in preserving thyroid function.

Table 2 Thyroid Function Tests in Case vs. Control Groups.
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Table 3 presents the prevalence of categorical thyroid dysfunction among the study participants. The results demonstrate a profound and highly significant disparity in thyroid status between BTM patients receiving iron chelation therapy (ICT) and those who were not.

Table 3 Prevalence of Thyroid Dysfunction in Case vs. Control Groups.
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The vast majority of patients in the control group (88%) exhibited some form of hypothyroidism. This was comprised of 45% with subclinical hypothyroidism and 43% with overt hypothyroidism. In stark contrast, only 22% of the ICT-treated patients were hypothyroid, with the overwhelming majority (78%) maintaining a euthyroid state. The differences in the prevalence of euthyroid status, subclinical hypothyroidism, and overt hypothyroidism between the two groups were all statistically significant (p < 0.001 for all categories).

Table 4 compares thyroid function parameters among beta-thalassemia major (BTM) patients receiving different iron chelation therapies. Table 4 findings suggest that the choice of chelator may differentially influence thyroid function, with deferasirox appearing to offer superior protection against thyroid dysfunction. The observed variations highlight the need for personalized chelation strategies in BTM management, particularly for patients at risk of endocrine complications. The statistically robust differences (all p < 0.001) underscore the clinical relevance of these findings for therapeutic decision-making.

Table 4 Thyroid Function Parameters by Iron Chelator Type.
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Table 5 presents a comprehensive comparison of liver and renal function parameters between beta-thalassemia major (BTM) patients receiving iron chelation therapy (ICT) and BTM controls. The results demonstrate highly significant differences (p < 0.001 for all parameters) in hepatic and renal biomarkers between the two groups. Table 4 findings collectively suggest that ICT may mitigate the multi-organ complications associated with chronic iron overload in BTM patients. The substantial differences across all measured parameters (p < 0.001) underscore the systemic benefits of chelation therapy beyond its primary role in managing iron burden.

Table 5 Liver and Renal Function Tests.
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Table 6 presents the correlation matrix examining relationships between ICT, thyroid function parameters, and serum ferritin levels in BTM patients. The analysis reveals several clinically significant associations. Table 5 findings suggest that iron overload severity directly impacts thyroid function, with higher ferritin levels associated with greater thyroid dysfunction. These correlation patterns provide important insights into the complex interplay between iron burden, chelation therapy, and endocrine function in thalassemia patients. The particularly strong ferritin-TSH association underscores the importance of aggressive iron control in preventing thyroid complications in this vulnerable population. The differential correlation strengths among thyroid parameters may reflect varying stages of subclinical dysfunction in the study cohort.

Table 6 Correlations Between Iron Chelation Therapy, Thyroid Parameters, and Ferritin.
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Table 7 presents the results of multivariate regression analysis examining predictors of TSH levels in BTM patients receiving iron chelation therapy. The model accounted for thyroid hormones, iron overload status, chelator type, and demographic factors, revealing several key findings. Table 6 findings highlight iron overload as the primary modifiable risk factor for thyroid dysfunction in this population, while suggesting that chelator selection may have limited independent impact on TSH regulation when adequate iron control is achieved.

Table 7 Multivariate Regression Coefficients for Predicting TSH Levels.
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Table 8 presents the ANCOVA results examining factors influencing free T4 levels in BTM patients, with particular focus on ICT type while controlling for potential confounders.

Table 8 ANCOVA Summary Table for Thyroid Function (Free T4).
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Table 9 presents the Pearson correlation coefficients with associated p-values, examining relationships between thyroid function parameters and iron status markers. The correlation matrix confirms the central role of iron burden in thyroid dysfunction while revealing differential sensitivity of various thyroid parameters to iron overload effects. These patterns provide important insights into the pathophysiology of iron-induced endocrine complications in thalassemia patients. The extremely high significance levels (all p < 0.001 for major correlations) and large effect sizes (particularly for ferritin-TSH relationship) underscore the clinical importance of these findings for monitoring and managing thyroid function in iron-overloaded patients. The differential correlation strengths among thyroid parameters may reflect their varying utility as early markers of iron-induced endocrine dysfunction.

Table 9 Pearson Correlation Coefficients with p-values.
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Discussion

This study evaluated the impact of iron chelation therapy (ICT) on thyroid function in patients with beta-thalassemia major (BTM) in Mardan, Pakistan. Our findings demonstrate that ICT significantly mitigates thyroid dysfunction, with deferasirox showing the most favorable thyroid profile (lowest TSH levels). Serum ferritin emerged as the strongest predictor of thyroid impairment, underscoring the critical role of iron overload in endocrine disruption13. Additionally, ICT was associated with preserved hepatic and renal function, highlighting its systemic benefits beyond iron control. These results align with the study’s objective of optimizing chelation strategies to reduce endocrine complications in BTM patients, particularly in resource-limited settings.

The observed protective effect of ICT on thyroid function is consistent with prior studies demonstrating that iron overload contributes to hypothyroidism in thalassemia patients14. However, our finding that deferasirox was associated with lower TSH levels contrasts with some earlier reports suggesting comparable efficacy among chelators15. This discrepancy may reflect differences in patient adherence, dosing regimens, or genetic factors influencing drug metabolism. The strong correlation between ferritin and TSH (r = 0.94, p < 0.001) reinforces the well-established link between iron burden and thyroid dysfunction16, though our multivariate analysis uniquely identifies ferritin as the sole independent predictor of TSH levels after adjusting for confounders.

Our findings are consistent with a growing body of evidence demonstrating a strong association between iron overload and thyroid dysfunction in patients with beta-thalassemia major. A systematic review and meta-analysis by Haghpanah et al. reported a pooled prevalence of hypothyroidism ranging from 6 to 30% in transfusion-dependent thalassemia, with serum ferritin identified as a major determinant of endocrine involvement4,17. Similarly, Singhal and Goyal observed significantly higher TSH levels and lower free T4 concentrations in hypothyroid thalassemia patients compared with euthyroid counterparts, reinforcing the dose-dependent toxic effect of iron deposition on thyroid tissue18,19. Ghosh and Chakrabarti further demonstrated a positive correlation between serum ferritin and TSH levels, suggesting that iron-mediated oxidative damage disrupts pituitary–thyroid axis regulation20. The extremely strong ferritin–TSH correlation observed in our cohort (r = 0.94, p < 0.001) aligns closely with these reports and highlights the critical importance of iron burden control in preventing thyroid dysfunction.

Emerging evidence suggests that the type of iron chelation therapy may influence endocrine outcomes beyond iron removal alone. Casale et al., in a large multicenter cohort study, reported a lower incidence of endocrine complications—including hypothyroidism—among patients receiving deferasirox compared with those on other chelation regimens, attributing this effect to improved adherence and sustained ferritin reduction21,22. Similarly, Premawardhena et al. highlighted that oral chelators, particularly deferasirox, achieve more consistent iron control compared with parenteral deferoxamine, which may translate into better long-term organ protection23,24. Although some studies have reported comparable thyroid outcomes across chelators, these discrepancies may reflect differences in sample size, treatment duration, or adherence patterns25,26. Our findings support the growing clinical perspective that effective and sustained iron chelation—rather than chelator choice alone—is the primary determinant of endocrine preservation, with deferasirox offering practical advantages in real-world settings through improved compliance and iron load reduction.

In 2020 at Umaid hospital, affiliated with S.N. Medical College, Jodhpur, children were tested for thyroid function and ferritin levels as part of the study. The study involved 112 participants. The number of euthyroid children was 82 (73.2%), while the number of hypothyroid children was 30 (26.2%). Euthyroid children had mean serum ferritin, serum TSH, serum free T4 and serum free T3 levels of 1975.4 + 706.2 (ng/ml), 3.23 + 0.93 (IU/ml), 12.8 + 2.3 (pmol/l) and 6.12 + 1.4 (pmol/l), respectively. Ferritin, TSH, free T4 and free T3 levels in a hypothyroid child were 2843.92 1095.22 (ng/ml), 7.05 1.91 (IU/ml), 10.55 2.0 (pmol/l) and 4.49 1.2 (pmol/l), respectively. Thyroid dysfunction is a common complication of regular blood transfusions in thalassemic patients. Iron overload plays a significant role in the development of thyroid dysfunction by influencing serum ferritin levels. An extensive endocrinological follow up, including annual screening, is essential for patients suffering from beta thalassemia major to achieve a good quality of life14.

The superior hepatic and renal outcomes in ICT-treated patients align with previous research showing that effective iron cheglation reduces end-organ damage27. However, our results extend these findings by demonstrating that thyroid protection may vary by chelator type, with deferasirox potentially offering additional endocrine benefits. This contrasts with a study that reported no significant differences in thyroid function across chelators, possibly due to smaller sample sizes or shorter follow-up periods28. Our larger cohort and rigorous statistical adjustments strengthen the validity of these comparative findings.

Clinical implications

These results have immediate clinical relevance for managing BTM in resource-limited settings. The strong ferritin-TSH association supports aggressive iron monitoring, with deferasirox emerging as a preferred option for patients at high risk of thyroid dysfunction. Given the high prevalence of BTM in Pakistan, these findings advocate for routine thyroid screening in thalassemia care protocols and personalized chelation strategies based on individual risk profiles.

Limitations and future directions

While this study provides valuable insights, several limitations should be considered when interpreting the results. Firstly, the cross-sectional design precludes the establishment of causal relationships between iron chelation therapy and thyroid outcomes; it can only identify associations. The single-center recruitment of participants may introduce potential selection bias, as our cohort might not be fully representative of the broader BTM population in Pakistan, particularly in terms of treatment adherence and access to care.

Although we used standardized laboratory techniques, the possibility of measurement variability in biomarkers like serum ferritin, which can be influenced by acute phase reactions, must be acknowledged. Furthermore, despite our efforts to control for key variables through multivariate regression, the influence of uncontrolled confounders cannot be entirely ruled out. Factors such as genetic predispositions, nutritional status (including iodine and selenium levels), precise cumulative iron load from transfusions, and detailed records of patient compliance with chelation regimens were not available and could potentially influence thyroid function.

Future research should employ prospective, longitudinal designs to track thyroid function over time and better establish causality. Multicenter studies encompassing diverse ethnic and socioeconomic groups would enhance the generalizability of the findings. Further investigation into the genetic and nutritional modifiers of iron-induced endocrine damage is also warranted to enable truly personalized treatment strategies for patients with beta-thalassemia major.

Conclusion

In conclusion, this cross-sectional analysis demonstrates a significant association between iron chelation therapy and improved thyroid function in patients with beta-thalassemia major. Our findings suggest that ICT, particularly with deferasirox, appears to be protective against thyroid dysfunction, with serum ferritin levels serving as a key biomarker for endocrine risk. While these results indicate a strong beneficial relationship, the cross-sectional nature of the study design confirms association rather than causation.

These findings underscore the clinical importance of tailored chelation strategies and systematic endocrine monitoring in thalassemia management. For regions like Pakistan, where BTM prevalence is high, integrating these insights into national treatment guidelines could significantly help in mitigating endocrine complications and improving patient outcomes. Longitudinal studies are needed to confirm these associations and establish causal relationships.