INTRODUCTION

Hemoglobinopathies are among the most prevalent inherited genetic disorders worldwide, characterized by either reduced synthesis or structural abnormalities of hemoglobin chains. ^{1, 2} Sickle cell disease (SCD) and thalassemia syndromes are the most common forms, leading to chronic anemia, end-organ damage, and significant healthcare burdens. ^{3, 4} In India, anemia is a major public health issue, especially in children, contributing to poor growth, cognitive delay, and increased morbidity. ^{5, 6}

According to the World Health Organization, about 5% of the global population are carriers of hemoglobinopathies, with the highest burden observed in regions such as sub-Saharan Africa, the Middle East, and South Asia. ^{2, 7} India alone harbors an estimated 30 million carriers, with a mean prevalence of 3.3%. ⁸ Beta-thalassemia trait incidence ranges from 3% to 17%, and the sickle cell gene prevalence may reach up to 44% in certain Indian regions, particularly among tribal communities. ^{9, 10} Maharashtra, with its diverse population, shows considerable variation in prevalence, with higher rates in tribal populations. ¹¹

Early diagnosis and intervention are vital for managing hemoglobinopathies. High-performance liquid chromatography (HPLC) has emerged as a sensitive and reliable tool for detecting abnormal hemoglobin variants, especially in children presenting with anemia. ^{12, 13} Cation-exchange HPLC offers rapid, automated, and accurate hemoglobin fractionation, making it ideal for large-scale screening. ¹⁴

Given the genetic and clinical significance of hemoglobinopathies in the pediatric population, especially in high-prevalence areas, this study was conducted to evaluate the prevalence and spectrum of hemoglobinopathies among anemic children in a tertiary care hospital using HPLC and other hematological parameters. The findings aim to inform early screening strategies, genetic counseling, and public health planning.

The objective of this study was to assess the prevalence of hemoglobinopathies and hemoglobin variants among anemic children admitted to a tertiary care teaching hospital. Additionally, the study aimed to analyze the demographic profile and clinical presentation of the study population, evaluate their complete blood count (CBC) parameters, and provide appropriate counselling to the parents of affected children. The goal of counseling was to promote awareness, facilitate early diagnosis, and help prevent future cases through family screening and genetic guidance.

MATERIALS AND METHODS

RESULTS

Of 516 anemic children (6 months–12 years), 278 (53.88%) were male and 238 (46.12%) females, with 227 (43.99%) aged 0.5–5 years, 195 (37.79%) 6–10 years, and 94 (18.22%) 11–12 years. Consanguinity was reported in 109 (21.12%), and 103 (19.96%) had prior transfusions; among 70 children with hemoglobinopathies, 21 (30%) had consanguineous parents (Table 1).

Pallor was the most common symptom (325, 62.98%), followed by fatigue (103, 19.96%), breathlessness (58, 11.24%), and fever (48, 9.30%). In the variant group (n=70), pallor occurred in 54% (vs. 62.98% overall, p=NS), fatigue in 19%, and fever in 6% (Table 1).

Hematological data (Table 2) showed Hb levels of 10.1–12 g/dL in 344 (66.66%), 6.1–9.0 g/dL in 111 (21.51%), and ≤6 g/dL in 61 (11.82%). RBC count was <4.7 ×10⁶/μl in 423 (81.97%), MCV 60–75 fL in 395 (76.55%), MCH <27 pg in 336 (65.12%), MCHC <32 g/dL in 239 (46.32%), RDW >14.5% in 296 (57.36%), and PCV <30% in 349 (67.64%). Reticulocyte counts were 0.5–2.5% in 452 (87.60%) and >2.5% in 64 (12.40%). HPLC identified hemoglobinopathies in 70 children (13.57%), predominantly sickle cell trait (29, 5.62%), sickle cell disease (17, 3.29%), β-thalassemia trait (13, 2.52%), and β-thalassemia major (7, 1.36%) (Table 3). Rarer variants included S-β thalassemia (2, 0.39%), heterozygous HbE (1, 0.19%), and SCT + HbD trait (1, 0.19%). Most cases (49, 70%) were aged 0.5–5 years, with 14% each in 6–10 and 11–12 years. Transfusions were required by 103 (19.96%) overall, with rates of 71.4% in β-thalassemia major, 41.2% in sickle cell disease, and 10.3% in sickle cell trait (Table 3).

Caste analysis revealed higher variant rates in Manelwarli (4/14, 28.57%), Navhi (5/21, 23.81%), Matang (2/9, 22.22%), Banjara (5/26, 19.23%), and Mahar (4/22, 18.18%) (Table 4).

Hematological parameters (Table 5) showed β-thalassemia major with the lowest Hb (4.97 ± 1.70 g/dL) and sickle cell disease with high HbS.

(69.8 ± 9.82%) and HbF (20.98 ± 1.85%). Variant vs. normal comparisons revealed lower Hb (8.46 ± 1.14 vs. 9.82 ± 2.56 g/dL, p=0.0102), higher MCV (72.63 ± 3.15 vs. 63.91 ± 6.9 fL, p<0.0001), and higher RDW (15.76 ± 1.38 vs. 14.77 ± 1.86, p=0.0226), with no significant differences in MCH (22.5 ± 2.1 vs. 23.1 ± 3.0 pg, p=0.35) or PCV (25.8 ± 4.2 vs. 26.3 ± 5.1%, p=0.42). Screening tests: The sickling test was positive in 85 cases (16.47%), of which 54% were confirmed to have hemoglobin variants on HPLC. The solubility test was positive in 82 cases (15.89%), with 56% confirmed as variants.

DISCUSSION

Hemoglobinopathies are among the most common monogenic disorders worldwide, particularly prevalent in developing countries like India where consanguinity and endogamy contribute significantly to their inheritance patterns.

This study aimed to evaluate the prevalence and spectrum of hemoglobinopathies in anemic children using high-performance liquid chromatography (HPLC), and to correlate clinical, hematological, and biochemical findings with hemoglobin variant types.

In the present study, the prevalence of hemoglobinopathies was found to be 13.57% among anemic children, which is consistent with findings from other Indian studies, such as Shah et al. ¹⁵ (2012) who reported 13.1%, and Verma et al. ¹⁶ (2011) who found similar rates in pediatric populations using HPLC. This moderate-to-high prevalence reinforces the importance of routine screening for hemoglobin variants in symptomatic and anemic children.

Sickle cell trait (5.62%) was the most frequently detected abnormality, followed by sickle cell disease (3.29%), and β-thalassemia trait (2.52%). This aligns with data from western India, especially tribal and semi-urban areas of Maharashtra, where the sickle gene frequency is high. The findings are comparable to studies conducted by Madan et al. ¹⁷ (2010) and Patel et al. (2013) ¹⁸ , which reported similar variant distribution.

The majority of variant hemoglobin cases (70%) were observed in the 0.5–5-year age group, which may reflect both the early onset of symptoms and better detection due to increased awareness and healthcare-seeking behavior. This early detection is vital for timely initiation of interventions such as regular monitoring, prophylaxis for infections, and genetic counseling.

Consanguinity was reported in 30% of the variant hemoglobinopathy cases. This is a well-established risk factor for autosomal recessive disorders like β-thalassemia and sickle cell disease. Our findings are in concordance with those of Choudhary et al. ¹⁹ (2015), who also highlighted a strong link between parental consanguinity and the inheritance of hemoglobinopathies.

In terms of clinical presentation, pallor was the most common symptom both in the overall population and in the variant subgroup, followed by fatigue and breathlessness. These findings reflect the typical features of chronic anemia due to ineffective erythropoiesis and hemolysis. Fever and failure to thrive were also frequent, especially in children with sickle cell disease and thalassemia major, reflecting the systemic impact of these conditions.

The hematological profiles showed distinct patterns between normal and variant groups. Children with hemoglobinopathies had significantly lower hemoglobin (mean: 8.46 g/dL) and higher MCV and RDW values. The elevated MCV in variant cases may be due to compensatory erythropoiesis and bone marrow hyperplasia, especially in thalassemia carriers. RDW, a marker of anisopoikilocytosis, was significantly raised in variant cases (p=0.0226), reflecting ineffective erythropoiesis and red cell heterogeneity. These findings are in agreement with previous studies by Jain et al. ²⁰ and Rangan et al. ²¹

Out of 103 children who required blood transfusions, the highest requirement was noted in β-thalassemia major (71.4%) and sickle cell disease (41.2%). No transfusions were required in cases with β-thalassemia trait or HbE heterozygosity. This supports the classification of β-thalassemia major and sickle cell disease as clinically severe forms requiring intensive supportive care, while trait forms remain largely asymptomatic.

The caste-wise distribution revealed a higher frequency of hemoglobin variants in certain scheduled and tribal communities such as Manelwarli, Navhi, Matang, and Banjara. This likely reflects the genetic pooling due to socio-cultural practices of endogamy. ²² These findings suggest that community-targeted screening programs may be highly effective in early identification and management.

HPLC proved to be a highly effective diagnostic tool, identifying abnormal hemoglobin fractions with precision. Among the screening tests, the sickling test, solubility test, and NESTROFT had moderate concordance with HPLC results, confirming variants in approximately 52–56% of cases. These conventional screening methods, although cost-effective, have limited sensitivity and specificity, underscoring the importance of confirmatory testing with HPLC. ²³

This study also reported detailed fractionation profiles for each hemoglobinopathy. For instance, sickle cell disease patients had high HbS and HbF, while β-thalassemia trait was marked by elevated HbA2. Such detailed profiling is essential for distinguishing between similar clinical entities like SCD, SCT, and S-β thalassemia.

Despite the strengths of this study, such as a sizeable sample and comprehensive analysis, limitations include its single-center design and exclusion of molecular confirmation techniques such as gene sequencing or ARMS-PCR. Additionally, neonatal cases and family studies were not included, which might have provided further insights into inheritance patterns.

CONCLUSIONS

This study highlights the significant burden of hemoglobinopathies among anemic children, with a prevalence rate of 13.57%, reinforcing the importance of early detection and intervention. Sickle cell trait, sickle cell disease, and β-thalassemia trait emerged as the most common variants, with a notable prevalence particularly in younger children and specific caste groups linked to consanguinity. These findings underscore the genetic and regional factors driving these disorders, emphasizing their relevance as a public health concern in India.

High-performance liquid chromatography (HPLC) demonstrated its superiority over traditional screening methods for accurate diagnosis and classification, supporting its integration into clinical practice. The severity of conditions like β-thalassemia major and sickle cell disease, marked by significant transfusion needs, further stresses the importance of timely management. Given these insights, community-based screening, public education, and genetic counseling are essential strategies to reduce this preventable burden, particularly in high-risk populations. A comprehensive approach combining advanced diagnostics, clinical vigilance, and preventive measures is vital to address the challenge of hemoglobinopathies effectively.

Disclosure

Funding: None

Conflict of Interest: None Declared

Author Contribution: All the authors involved in study have contributed equally at all stages of work

Acknowledgements: I would like to express my appreciation to all those who have supported and contributed to the completion of this project