Inherited hemoglobinopathies, including sickle cell anemia (SCA) and β-thalassemia, are among the most prevalent monogenic disorders worldwide, significantly contributing to morbidity and healthcare burden, particularly in regions where these conditions are endemic¹. Both disorders arise due to genetic mutations affecting hemoglobin production, leading to chronic hemolysis, ineffective erythropoiesis, and a wide spectrum of clinical manifestations ranging from mild anemia to life-threatening complications². A key factor influencing the clinical severity of SCA and β-thalassemia is the level of fetal hemoglobin (HbF), which is encoded by the HBG1 and HBG2 genes. HbF is the predominant hemoglobin during fetal life, gradually replaced by adult hemoglobin (HbA) after birth. However, in certain individuals, persistent expression of HbF into adulthood modifies disease severity³. HbF reduces sickling in SCA by inhibiting polymerization of sickle hemoglobin (HbS), thereby decreasing vaso-occlusive crises, hemolysis, and end-organ damage. Similarly, in β-thalassemia, elevated HbF partially compensates for defective HbA synthesis, alleviating anemia and reducing the need for frequent blood transfusions⁴.  Blood transfusion remains a cornerstone of management in severe cases of SCA and β-thalassemia, helping to maintain adequate hemoglobin levels, prevents complications, and improves quality of life. However, transfusion dependency varies widely among patients, influenced by factors such as baseline HbF levels, degree of hemolysis, and overall disease burden. While individuals with higher endogenous HbF often require fewer transfusions, those with low HbF levels may experience more severe anemia, necessitating regular transfusions to prevent complications such as growth retardation, iron overload, and cardiac dysfunction⁵. Understanding these associations is crucial for guiding personalized treatment approaches, including the use of HbF-inducing agents like hydroxyurea or emerging gene-editing therapies. High-performance liquid chromatography (HPLC) is an established, reliable diagnostic tool for detecting abnormal hemoglobin variants and quantifying HbF levels in patients with hemoglobinopathies⁶. Despite extensive research on HbF’s role in disease modification, the precise relationship between HbF levels, hemoglobinopathy types, and blood transfusion requirements remains an area of ongoing investigation. This study aims to compare HbF levels across different hemoglobinopathy groups, including sickle cell anemia (SCA), β-thalassemia major (BTM), β-thalassemia trait (BTT), sickle cell trait (SCT), and sickle-thalassemia trait (STT) and to analyze the association between HbF levels and blood transfusion requirement in Hemoglobinopathy patients.

Study Design: This was a cross-sectional, hospital-based study conducted in the Department of Pathology at Gandhi Medical College and Associated Hospitals, Bhopal, from August 1, 2022, to January 31, 2024.

Study Population: The study included patients diagnosed with various hemoglobinopathies, including β-thalassemia major (BTM), β-thalassemia trait (BTT), sickle cell anemia (SCA), sickle cell trait (SCT), and sickle-thalassemia trait (STT), who consented to participate. The primary focus was to evaluate HbF levels across these groups and analyze their association with blood transfusion requirements.

Inclusion Criteria

• Patients diagnosed with β-thalassemia major, β-thalassemia trait, sickle cell anemia, sickle cell trait, or sickle-thalassemia trait.
• Patients who provided informed consent.

Exclusion Criteria

• Patients with a history of blood transfusion in the past three months to avoid transient alterations in HbF levels.
• Patients with other hematological disorders that could affect HbF levels.
• Patients who declined to participate in the study.

Sample Size: A total of 130 patients with hemoglobinopathies were included in the study, recruited from the hospital during the study period.

Ethical Considerations: The study was approved by the Institutional Ethics Committee of Gandhi Medical College, Bhopal. Informed consent was obtained from all participants, and confidentiality of patient data was maintained.

Sample Collection: A 2 mL venous blood sample was collected from each participant in a K₃EDTA vacutainer during routine investigations or at their convenience, ensuring no additional cost to the patients.

Laboratory Analysis: Hemoglobin analysis was performed using High-Performance Liquid Chromatography (HPLC) to measure fetal hemoglobin (HbF) levels in all participants. Peripheral blood smears were prepared, stained with Leishman’s stain, and examined microscopically for morphological assessment of red blood cells.The sickling test was conducted for sickle cell disease (SCD) and sickle cell trait (SCT) patients, when necessary, to confirm diagnosis.

Statistical Analysis

Data were entered into Microsoft Excel and analyzed using SPSS software.Descriptive statistics (mean, standard deviation, and percentages) were used to summarize HbF levels and transfusion requirements across different hemoglobinopathy groups.The association between HbF levels and blood transfusion requirements was assessed using the independent t-test, comparing mean HbF levels in transfusion-dependent and non-transfusion-dependent patients.Comparisons between different hemoglobinopathy groups were conducted using one-way ANOVA. A p-value < 0.05 was considered statistically significant.

Table 1 compares the frequency of clinical complaints across various diagnostic groups, including Beta Thalassemia Major (BTM), Beta Thalassemia Trait (BTT), Sickle Cell Disease (SCD), Sickle Cell Trait (SCT), and Sickle Cell-Thalassemia Trait (STT).

Table 1: Distribution of Clinical Complaints Among Different Hemoglobinopathy Groups

Fever and weakness were the most common complaints, with SCT patients reporting the highest prevalence

Table 2: Hemoglobin F (HbF) Levels Across Different Hemoglobinopathy Groups

A statistically significant difference (P < 0.001) was observed in HbF levels across the groups. BTM had the highest HbF levels (Mean = 86.95%), followed by SCD (Mean = 13.72%) and STT (Mean = 8.18%), while normal individuals had the lowest levels (Mean = 1.77%).

Table 3: HbF Levels in Beta Thalassemia Major vs. Beta Thalassemia Trait

A highly significant difference (P < 0.001) was noted between BTM and BTT, emphasizing the role of HbF in disease severity modulation.

Table 4: HbF Levels in Sickle Cell Disorders

SCD patients exhibited significantly higher HbF levels compared to SCT individuals (P < 0.001), reinforcing the protective role of HbF in reducing disease severity.

Table 5: Association of HbF Levels with Blood Transfusion Requirement

Patients requiring blood transfusions had significantly higher HbF levels (Mean = 24.5%) compared to those not requiring transfusions (Mean = 2.94%) (P < 0.001), suggesting that higher HbF may delay transfusion dependence

Fetal hemoglobin (HbF) plays a pivotal role in modulating the clinical severity of hemoglobinopathies, particularly sickle cell disease (SCD) and beta-thalassemia. HbF inhibits the polymerization of deoxygenated sickle hemoglobin (HbS), thereby reducing erythrocyte sickling, hemolysis, and vaso-occlusive complications in SCD patients. Additionally, in Beta Thalassemia Major (BTM), where there is an absence of functional adult hemoglobin (HbA), HbF serves as the primary oxygen-carrying molecule. Our study demonstrated that individuals with BTM exhibited markedly elevated HbF levels (Mean = 86.95%), while SCD patients also had higher HbF levels (Mean = 13.72%), reinforcing the protective role of HbF in reducing disease severity. These findings are consistent with previous studies, such as those by Steinberg et al. (2014), who reported that increased HbF levels are associated with reduced frequency and severity of vaso-occlusive crises, lower risk of stroke, and decreased incidence of acute chest syndrome in SCD patients.⁷

The role of HbF in modifying disease severity has been well documented in prior research. Kulozik et al. (1987) demonstrated that the Asian βS haplotype, commonly found in Indian sickle cell patients, is associated with higher HbF levels (mean 15–25%), leading to a milder clinical phenotype compared to other sickle cell populations⁸. This aligns with our study, where SCD patients showed HbF elevation, though with variability in levels, likely due to underlying genetic modifiers. Akinsheyi et al. (2011) similarly reported that HbF levels were significantly higher in SCD patients (19.63%) and BTM patients (51.62%), highlighting that while elevated HbF can reduce symptoms in SCD, it cannot fully compensate for the severe anemia seen in β-thalassemia major⁹.

A major contributor to HbF variability among patients is the presence of genetic modifiers, particularly BCL11A, HBS1L-MYB, and KLF1 polymorphisms. These genetic regulators play a crucial role in fetal-to-adult hemoglobin switching and can significantly influence HbF levels. Thein et al. (2017) identified these genetic variants as major determinants of HbF levels and disease severity in hemoglobinopathies¹⁰. Nongbri et al. (2017) also emphasized the role of β-globin gene haplotypes in modulating HbF levels among Indian sickle cell patients, further underscoring the importance of genetic background in disease heterogeneity¹¹. Given this genetic influence, screening for HbF-associated polymorphisms may be useful for predicting disease severity and tailoring individualized treatment strategies.

In addition to genetic regulation, another key determinant of disease severity is F-cell distribution, referring to the proportion of red blood cells that contain HbF. Although some patients have high overall HbF levels, an uneven distribution of HbF among red blood cells can lead to continued sickling and disease complications. Steinberg and Rodgers emphasized that F-cell proportion is a better predictor of disease severity than absolute HbF levels, as only a uniform distribution of HbF can effectively prevent erythrocyte sickling⁷. Our study supports this observation, as some patients with similar HbF levels exhibited varying clinical severities, suggesting that HbF distribution should be considered in disease prognosis. Future research incorporating flow cytometry-based F-cell quantification could provide deeper insights into the role of HbF distribution in modifying clinical outcomes.

Beyond genetic and cellular mechanisms, our study highlights the therapeutic potential of HbF induction in managing hemoglobinopathies. Hydroxyurea is the most widely used HbF-inducing drug, known to increase HbF levels and reduce SCD complications. Sankaran et al. (2015) demonstrated that hydroxyurea therapy leads to a significant elevation in HbF levels, decreasing pain crises, hospitalizations, and transfusion requirements¹². However, the response to hydroxyurea varies among patients, likely due to genetic polymorphisms influencing drug metabolism and HbF induction. Thus, pharmacogenetic testing may help optimize hydroxyurea dosing for individual patients. Additionally, newer HbF-inducing agents, such as decitabine, butyrate derivatives, and histone deacetylase inhibitors, are under investigation and may offer promising alternatives for patients with inadequate response to hydroxyurea.

The protective role of HbF in β-thalassemia is also well documented. Patients with β-thalassemia intermedia who have naturally high HbF levels often exhibit a milder clinical phenotype and lower transfusion requirements. Galanello et al. (2009) reported that β-thalassemia patients with higher HbF levels experience less hemolysis, fewer complications, and reduced transfusion dependency, further supporting the therapeutic importance of HbF elevation¹³. Similarly, Aleluia MM (2017) found that SCD patients with the Senegal haplotype, characterized by high HbF levels, experienced significantly milder disease symptoms, reinforcing the genetic basis of HbF variability and its impact on clinical severity¹⁴. These findings highlight the potential benefits of HbF-inducing therapies in both SCD and β-thalassemia, suggesting that patients with low endogenous HbF may benefit from early intervention with HbF-modulating drugs.

Despite its strengths, our study has certain limitations that should be acknowledged. The cross-sectional design limits our ability to establish causal relationships between HbF levels and disease severity. Additionally, our sample size, while representative, may not fully capture the genetic diversity of hemoglobinopathy patients, potentially affecting the generalizability of our findings. Future research should focus on larger, multi-center studies with longitudinal follow-ups to better understand the long-term impact of HbF levels on clinical outcomes. Moreover, investigating co-inherited genetic modifiers, such as alpha-thalassemia, could provide a more comprehensive picture of disease variability, as co-inheritance of α-thalassemia with SCD has been shown to modify disease severity by altering red cell indices and hemolysis rates.

Furthermore, while our study primarily focused on HbF levels, future research should explore additional biomarkers, such as erythropoietin levels, reticulocyte counts, and hemolysis markers, to gain a more holistic understanding of disease pathophysiology. Another area of interest is the impact of environmental and lifestyle factors, such as nutrition, infection, and inflammation, on HbF expression, as these factors may contribute to inter-individual variability in disease severity.

Our study confirms that HbF levels vary significantly across hemoglobinopathy subtypes, influencing disease severity and transfusion dependency. Higher HbF levels in SCD were associated with reduced clinical severity, while in BTM, despite elevated HbF, transfusion dependence persisted, indicating that HbF alone does not fully compensate for ineffective erythropoiesis. Patients requiring blood transfusions had significantly higher HbF levels, suggesting that disease severity is influenced by multiple factors beyond HbF concentration alone. HbF-inducing therapies, such as hydroxyurea, may help reduce complications in SCD, but their effectiveness varies among patients. Future research should focus on longitudinal studies and alternative therapeutic strategies to improve disease management and patient outcomes.

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