Thalassemia and sickle cell disease are two of the most common inherited hemoglobin disorders worldwide, causing significant morbidity and mortality. These conditions are characterized by abnormalities in the structure or production of hemoglobin, the molecule in red blood cells that delivers oxygen to cells throughout the body. Despite their prevalence, distinguishing between these diseases can be challenging, as they share overlapping clinical features but require different management strategies.^[1][2]

Thalassemia is primarily caused by mutations that reduce the synthesis of one of the two subunits of hemoglobin—alpha or beta. Depending on which gene is affected, the disease manifests as alpha or beta thalassemia. Patients with thalassemia major, the most severe form, often require regular blood transfusions and chelation therapy to manage iron overload.^[3][4]

Sickle cell disease, on the other hand, results from a mutation in the beta-globin gene that leads to the production of abnormal hemoglobin S (HbS). Under low oxygen conditions, HbS polymers cause red blood cells to become rigid and sickle-shaped, leading to vascular occlusion and ischemic damage.^[5][6]

The clinical presentation of both diseases includes symptoms of anemia, jaundice, and splenomegaly, and both can lead to severe complications if not managed appropriately. This underscores the importance of accurate and early diagnosis. Laboratory methods for diagnosis include complete blood counts, hemoglobin electrophoresis, and molecular testing, which provide insights into hemoglobin production and the presence of abnormal hemoglobin variants.^[7][8]

Aim

To evaluate and compare the hemoglobin variants in HPLC from patients with thalassemia and sickle cell disease.

 Objectives

1. To identify the specific hemoglobin variants present in HPLC of patients diagnosed with thalassemia.
2. To determine the hemoglobin variants in HPLC of patients diagnosed with sickle cell disease.
3. To compare the hemoglobin variants between thalassemia and sickle cell disease to aid in differential diagnosis.

Source of Data

Data were collected from patients diagnosed with either thalassemia or sickle cell disease, presenting at the hematology outpatient clinic of our tertiary care center.

Study Design

This was a cross-sectional observational study.

Study Location

The study was conducted at the Hematology Department of the Tertiary College Hospital.

Study Duration

The study was carried out from January 2024 to December 2024.

Sample Size

A total of 80 patients were included in the study, with 40 patients diagnosed with thalassemia and 40 with sickle cell disease.

Inclusion Criteria

Patients of any age and gender diagnosed with thalassemia or sickle cell disease based on previous hemoglobin (Hb) electrophoresis and molecular testing were included.

Exclusion Criteria

Patients with co-existing other major hematological disorders, those who had received a blood transfusion within the last three months, and those with incomplete medical records were excluded.

Procedure and Methodology

Blood samples were collected by venipuncture following standard aseptic techniques. Blood samples were processed for HPLC on BioRad Variant II Hemoglobin Testing Analyzer and reports were assessed to identify any specific hemoglobin variants.

Sample Processing

HPLC reports were assessed by experienced hematopathologists who were blinded to the clinical diagnosis. The presence of specific hemoglobin variants was recorded.

Statistical Methods

Data were analyzed using the SPSS software version 22.0. Descriptive statistics such as means and standard deviations were used to summarize continuous variables, and frequencies and percentages were used for categorical variables. Chi-square tests were employed to compare the prevalence of hemoglobin variants between thalassemia and sickle cell disease groups.

Data Collection

Data collection involved recording detailed demographic and clinical profiles of the patients, including age, gender, and diagnosis, along with the laboratory findings from HPLC reports.

Table 1: Evaluation and Comparison of Hemoglobin Variants in Thalassemia vs. Sickle Cell Disease

Table 1 provides a detailed comparison of mean hemoglobin variants between patients with thalassemia and those with sickle cell disease. The data reveal significant differences between the two groups across all measured variants. Hemoglobin A levels were slightly higher in thalassemia patients (9.4 g/dL) compared to sickle cell patients (8.6 g/dL), with a statistically significant difference (P = 0.004) and a confidence interval for the mean difference ranging from 0.4 to 1.2 g/dL. Notably, Hemoglobin S showed a dramatic contrast, being nearly negligible in thalassemia (1.7%) compared to a predominant presence in sickle cell disease (78.3%), with a highly significant P value (<0.001) and a large confidence interval difference (73.5 to 83.1%). Hemoglobin F and A2 also showed significant differences, underscoring distinct pathophysiological manifestations in these conditions.

Table 2: Specific Hemoglobin Variants in Thalassemia

In Table 2, the distribution of specific hemoglobin variants within the thalassemia patient group is detailed. Half of the patients exhibited Hemoglobin A and A2 (each at 50%), while Hemoglobin F was present in 40% of the patients. Lesser prevalent variants included Hemoglobin H (10%) and Bart's (15%). All variants showed statistically significant proportions within the study group, with P values ranging from 0.019 to 0.045, indicating reliable detection within this clinical setting.

Table 3: Specific Hemoglobin Variants in Sickle Cell Disease

Table 3 focuses on the distribution of hemoglobin variants among sickle cell disease patients. Hemoglobin S was the most prevalent, found in 50% of the patients, followed closely by Hemoglobin F at 45%. Hemoglobin A2 was seen in 15% of the cases, while Hemoglobin A was rare, found in only 5% of the patients. The statistical analysis provided (P values from 0.001 to 0.050) confirms the significant presence of these variants, defining the typical profile of sickle cell disease.

Table 4: Comparison of Hemoglobin Variants Between Thalassemia and Sickle Cell Disease

Table 4 contrasts the prevalence of hemoglobin variants between thalassemia and sickle cell disease, highlighting the differences that may assist in differential diagnosis. Hemoglobin A is predominantly found in thalassemia patients (50%) compared to sickle cell patients (5%), and Hemoglobin S is more typical of sickle cell disease (50%) than thalassemia (10%). Both Hemoglobin F and A2 display differing prevalences that are statistically significant, helping delineate the two disorders in a clinical setting. These differences are quantified with P values indicating strong statistical significance and confidence intervals that underscore the disparities between these two hemoglobinopathies.

Table 1: Evaluation and Comparison of Hemoglobin Variants in Thalassemia vs. Sickle Cell Disease

The differences in hemoglobin variants observed in our study are significant and align with established findings in hematological research. Our results show a pronounced prevalence of Hemoglobin S in sickle cell disease, which is a hallmark of the disease due to the mutation in the HBB gene resulting in abnormal hemoglobin that polymerizes under hypoxic conditions Adeyemo T et al.(2014)^[9]. This finding is consistent with the high percentages reported in literature, emphasizing the pathophysiological distinction from thalassemia, where Hemoglobin A predominates Lee YK et al.(2019)^[10]. The presence of higher Hemoglobin F in thalassemia patients compared to those with sickle cell disease supports existing research suggesting its role in ameliorating the severity of these disorders by inhibiting sickle hemoglobin polymerization and compensating for reduced beta-globin chain synthesis in beta-thalassemia Mohamad AS et al.(2018)^[11].

Table 2: Specific Hemoglobin Variants in Thalassemia

The distribution of hemoglobin variants such as Hb A, Hb F, and Hb A2 within thalassemia patients is well-documented Barrera‐Reyes PK et al.(2019)^[12]. The high frequency of Hb A and Hb A2 in our study reflects the typical pattern seen in beta-thalassemia carriers, where Hb A2 levels are elevated as a compensatory mechanism for the reduced beta-chain production. The presence of Hb H and Hb Bart's further corroborates with alpha-thalassemia, where gene deletions lead to excess unmatched beta globin chains forming beta4 tetramers (Hb H) and gamma4 tetramers (Hb Bart's) in more severe forms McGann PT et al.(2017)^[13].

Table 3: Specific Hemoglobin Variants in Sickle Cell Disease

Our findings of the predominance of Hb S in sickle cell disease patients are consistent with global observational studies Fonseca SF et al.(2015)^[14]. The significant presence of Hb F in these patients also aligns with therapeutic targets of hydroxyurea, which increases Hb F production as a treatment modality in sickle cell disease, demonstrating its protective effect against sickling episodes Franco E et al.(2024)^[15].

Table 4: Comparison of Hemoglobin Variants Between Thalassemia and Sickle Cell Disease

The comparative analysis between thalassemia and sickle cell disease reveals distinct profiles, crucial for differential diagnosis. The marked difference in Hb S and Hb A levels between the diseases reflects their underlying genetic differences and helps in guiding appropriate treatment strategies. These findings are in line with previous research which highlights the importance of characterizing hemoglobin profiles for accurate diagnosis and management of hemoglobinopathies Serjeant GR. (2022)^[16].

The cross sectional study has provided critical insights into the distinct hemoglobin profiles characteristic of thalassemia and sickle cell disease, two major hemoglobinopathies with significant global health impact. Our findings delineate clear differences in hemoglobin variants between these two disorders, which are crucial for accurate diagnosis and subsequent management.

In thalassemia, the elevated levels of Hemoglobin A and A2 and the presence of Hb H and Hb Bart's underscore the disease's nature of impaired hemoglobin production, predominantly due to beta-globin chain abnormalities or alpha-globin gene deletions. Conversely, the predominance of Hemoglobin S in sickle cell disease highlights the pathological consequence of the beta-globin gene mutation leading to abnormal hemoglobin polymerization under hypoxic conditions. Additionally, the comparative analysis of Hemoglobin F levels between the diseases illustrates its role in modulating disease severity and offers a therapeutic target in sickle cell disease management.

This study not only reinforces the importance of specific hemoglobin variant analysis in the differential diagnosis of hemoglobinopathies but also emphasizes the potential for targeted therapies that exploit these differences. The significant statistical associations and confidence intervals reported provide robust support for using these biomarkers in clinical settings to guide treatment strategies effectively.

Overall, the detailed evaluation of hemoglobin variants through high profile liquid chromatography presented in this study contributes to the body of knowledge necessary for advancing diagnostic accuracy and improving patient outcomes in populations affected by these challenging genetic blood disorders. Future research should continue to focus on expanding the understanding of hemoglobin variants in diverse populations and exploring innovative therapeutic approaches based on these findings.

1. Cross-Sectional Design: As a cross-sectional study, the research captures data at a single point in time, which limits the ability to ascertain causality or track changes in hemoglobin variants over time. Longitudinal studies are needed to observe the progression of these diseases and the long-term effects of various treatments.
2. Sample Size: With a total sample size of 80, split equally between patients with thalassemia and sickle cell disease, the study may not have adequate power to detect small differences in less common hemoglobin variants. A larger sample size would improve the statistical power and generalizability of the findings.
3. Geographical Limitation: The study was conducted at a single tertiary care center, which may limit the generalizability of the findings to other populations with different ethnic backgrounds and genetic predispositions to hemoglobinopathies.
4. Selection Bias: The inclusion of patients based on a clinical diagnosis and previous hemoglobin electrophoresis might introduce selection bias, affecting the representation of the entire spectrum of each disease.
5. Exclusion Criteria: Excluding patients who had received a blood transfusion in the three months prior to the study might have excluded a subgroup of patients with severe forms of the diseases, potentially skewing the severity and range of hemoglobin variants observed.
6. Confounding Variables: The study did not control for several potential confounding variables such as age, gender, nutritional status, or the presence of other medical conditions, which could influence hemoglobin levels and variant expression

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