SPECTRUM AND DIAGNOSTIC CHALLENGES OF HEMOGLOBINOPATHIES: AN HPLC-BASED STUDY FROM A TERTIARY CARE CENTRE

Hemoglobinopathies and thalassemias collectively represent the most prevalent monogenic disorders globally, imposing a profound public-health burden, particularly in low- and middle-income countries.1,2 Approximately 7% of the world's population carries a pathological haemoglobin gene, and an estimated 300,000–400,000 affected children are born annually.3,4 In India, the epidemiological landscape is shaped by consanguineous marriages, caste endogamy, and marked regional variation: the carrier frequency for β-thalassemia trait ranges from 3–4% nationally, reaching 17% in certain communities, while sickle cell anaemia prevalence varies from 1% in the general population to as high as 35% in some tribal groups.5,6,7

Haemoglobin is a tetrameric protein composed of two α-globin and two non-α-globin chains, each enwrapping a haem moiety.8 Disorders of haemoglobin are classified as: (i) structural (qualitative) variants—such as HbS (Glu→Val, β6), HbC (Glu→Lys, β6), HbD-Punjab (Glu→Gln, β121), and HbE (Glu→Lys, β26)—which arise from single amino-acid substitutions; and (ii) thalassemias (quantitative variants)—α- and β-thalassemias—resulting from reduced or absent globin-chain synthesis due to gene deletions or mutations in regulatory regions.8,9,10

 

High-performance liquid chromatography (HPLC) using cation-exchange chromatography is currently the gold-standard screening and diagnostic tool for hemoglobinopathies. The Bio-Rad Variant™ D10 system separates haemoglobin fractions—including HbA, HbA₂, HbF, and structural variants—by differential affinity to the ion-exchange column, and quantifies them as area percentages. The technique offers sensitivity exceeding 95%, specificity >99%, high throughput, and the ability to simultaneously detect rare variants.11,12

 

Despite HPLC's superiority, diagnostic pitfalls arise from overlapping retention windows, silent carriers, delta-beta thalassemia, hereditary persistence of foetal haemoglobin (HPFH), and novel mutations that generate unusual or unidentified peaks.13,14 Correlation with CBC indices, peripheral blood smear morphology, sickling tests, and molecular analysis is indispensable to resolve such ambiguities.

 

The tertiary paediatric centre (Niloufer Hospital, Hyderabad) serves a large, ethnically heterogeneous catchment population from Telangana and surrounding states, providing an ideal setting to characterise the spectrum of hemoglobinopathies. This retrospective study, therefore, aimed to: (1) determine the frequency and spectrum of hemoglobinopathies in paediatric patients referred for HPLC; (2) correlate HPLC findings with haematological and clinical parameters; and (3) highlight diagnostic challenges encountered during the study period.

 

Overall Distribution of Haemoglobin Patterns

Of the 1077 paediatric samples analysed, 847 (78.65%) showed normal haemoglobin patterns and 230 (21.35%) demonstrated abnormal patterns. The distribution of all haemoglobin types is summarised in Table 1 and illustrated in Figure 1.

 

Table 1: Overall Distribution of Haemoglobin Patterns (N=1077)

 

 

Figure 1. Distribution of haemoglobin patterns in 1077 paediatric samples by HPLC (N=1077).

Spectrum of Abnormal Haemoglobin Variants

β-Thalassemia trait was the most common abnormality, accounting for 116 cases (10.77% of total; 50.43% of abnormal samples). HPLC characteristically showed HbA₂ 3.5–8.0% with elevated HbA₂ peak and reduced HbA, correlating with microcytic hypochromic red cell indices (mean MCV 65.2 ± 7.4 fL, MCH 20.1 ± 3.8 pg). Unknown HPLC peaks were identified in 39 cases (3.62%), warranting further molecular work-up. Sickle cell trait was observed in 17 cases (1.58%), and homozygous sickle cell anaemia (HbSS) in 16 cases (1.49%). Details of abnormal variants are presented in Figure 2.

 

Figure 2. Proportional distribution of abnormal haemoglobin variants among 230 affected samples.

Haematological Parameters in Major Diagnostic Groups

Mean haematological and HPLC fraction data across the principal diagnostic categories are presented in Table 2 and Figure 3.

 

Table 2: Mean Haematological Indices and HPLC Fractions by Diagnostic Category (Mean ± SD)

 

Figure 3. Grouped bar chart of mean HPLC fractions (HbA, HbA₂, HbF, HbS) across major diagnostic categories.

HPLC Patterns of Key Haemoglobin Variants

The characteristic HPLC retention-time patterns and diagnostic cut-offs for the main variants identified in this study are summarised in Table 3 and visualised as a diagnostic heatmap in Figure 4.

 

Peripheral Smear Correlation

Peripheral smear findings supported HPLC interpretation in the majority of cases. Microcytosis with target cells was consistently observed in β-thalassemia trait and HbE trait; marked anisopoikilocytosis with nucleated RBCs and polychromasia was characteristic of β-thalassemia major/intermedia (Table 4). Sickled erythrocytes with polychromasia and Howell–Jolly bodies were identified in all 16 cases of homozygous sickle cell anaemia; target cells and occasional sickled cells were seen in sickle cell trait.

 

Table 3: Characteristic HPLC Profiles of Key Haemoglobin Variants (Bio-Rad D10 System)

 

Sickling Test Results

Sodium metabisulphite sickling test was performed in 33 patients with HPLC peaks in the S-window. All 16 cases of homozygous sickle cell anaemia (HbSS) showed strongly positive sickling. Of 17 sickle cell trait cases, 15 (88.2%) were positive; 2 (11.8%) showed a delayed positive result at 120 minutes, likely reflecting lower concentrations of HbS. In one case with an S-window peak and negative sickling, further work-up revealed HbD-Punjab, confirming that co-elution of HbD with HbS on D10 requires the sickling test for definitive differentiation. The compound heterozygous HbS+HbD case showed a positive sickling test with a complex HPLC pattern.

 

 

 

Figure 4. Diagnostic HPLC pattern heatmap showing characteristic haemoglobin fraction profiles for 10 variant types.

 

Table 4: Peripheral Blood Smear Morphology by Haemoglobin Condition

 

Molecular Findings

Molecular testing was performed in  three children with severe transfusion-dependent anaemia showed homozygous c.92+5G>C (IVS-I-5 G>C) mutation in the HBB gene, confirming β-thalassemia major  a splice-site mutation that leads to aberrant pre-mRNA processing and severely reduced β-globin production. This mutation is among the most common severe β-thalassemia mutations in South Asia.

Molecular results are summarised in Table 5.

 

 

Table 5: Summary of Molecular Analysis Findings

 

Comparison with Published Indian Studies

The prevalence rates in the present study were compared with published Indian HPLC-based paediatric and general population studies. As depicted in Figure 5, the β-thalassemia trait prevalence (10.77%) in our series is consistent with values reported from comparable tertiary centres, though slightly lower than some high-prevalence community studies.

 

 

 

Figure 5. Comparative prevalence of major haemoglobin variants (β-thalassemia trait and sickle cell trait) across published Indian HPLC studies.

The present study documents the spectrum of hemoglobinopathies in a large paediatric cohort (n=1077) at a tertiary care centre in Telangana using a comprehensive diagnostic workflow. The overall prevalence of abnormal haemoglobin patterns was 21.35%, highlighting the significant burden of these disorders in the catchment population. This figure is broadly concordant with estimates from comparable Indian tertiary-care studies, which report abnormal HPLC patterns in 15–35% of paediatric samples.1,2,15

 

β-Thalassemia Trait

β-Thalassemia trait was the predominant abnormality (116 cases, 10.77%), consistent with its reported prevalence of 3–17% in various Indian populations.5,6 Osmania Medical College and Niloufer Hospital serve Hyderabad’s multiethnic population, which includes communities such as Muslims, Brahmins, and certain tribal groups  known to have elevated carrier frequencies due to consanguineous marriages and endogamy.7 The HPLC hallmark of β-thalassemia trait is HbA₂ >3.5% with concurrent microcytosis (MCV <80 fL) and hypochromia (MCH <27 pg).12 One diagnostic challenge is the co-existence of iron-deficiency anaemia (IDA) with β-thalassemia trait, which can reduce HbA₂ into the borderline range (3.2–3.5%), leading to under-diagnosis.13 In our study, iron studies were not uniformly available; this limitation may have resulted in a few missed borderline diagnoses. Concurrent iron therapy and ferritin estimation is therefore recommended before HPLC-based screening.

 

Sickle Cell Disorders

Sickle cell trait (HbAS) was identified in 17 cases (1.58%) and homozygous sickle cell anaemia (HbSS) in 16 cases (1.49%). While these rates may appear low compared to Vidarbha or Chhattisgarh  regions with >20% HbS carrier frequency  Hyderabad’s mixed urban population yields intermediate values.16,17 The sodium metabisulphite sickling test remained a cost-effective and reliable adjunct: all 16 HbSS cases were strongly positive, and 15 of 17 trait cases tested positive. Notably, in one case with an HPLC peak in the S-window and a negative sickling test, HbD-Punjab was confirmed by molecular analysis. This underscores the fundamental principle that the sickling test is mandatory whenever an S-window peak is detected on HPLC, as HbD-Punjab co-elutes with HbS on the D10 system at identical retention times.18

The compound heterozygous HbS+HbD-Punjab (1 case) presented with a severe sickle cell phenotype similar to HbSS disease, characterised by positive sickling, splenomegaly, and haemolytic anaemia. Published literature confirms that HbS/HbD-Punjab compound heterozygotes may exhibit clinical severity comparable to HbSS, partly because HbD-Punjab does not inhibit HbS polymerisation.19

 

β-Thalassemia Major/Intermedia

β-Thalassemia major and intermedia accounted for 10 cases (0.93%). Molecularly, three of these children were confirmed to carry the homozygous c.92+5G>C (IVS-I-5 G>C) mutation in the HBB gene. This splice-site mutation disrupts normal pre-mRNA splicing, activating a cryptic splice site within intron 1, leading to reduced (or absent) functional β-globin chain production and consequent severe transfusion-dependent anaemia.20 The IVS-I-5 G>C mutation is one of the four most prevalent severe β-thalassemia mutations in South Asia (alongside IVS-I-1 G>A, codon 8/9 +G, and codon 41/42 −TTCT), accounting for approximately 15–18% of Indian β-thalassemia alleles.20,21

 

HPLC in β-thalassemia major characteristically shows a very high HbF fraction (>60%), near-absent HbA, and mildly elevated HbA₂. The peripheral smear revealed severe anisopoikilocytosis, polychromasia, nucleated RBCs, and tear-drop cells. All three confirmed cases were transfusion-naïve at initial presentation and were subsequently enrolled in a regular transfusion and chelation programme.

 

HPFH and δβ-Thalassemia

Seven cases (0.65%) showed raised HbF with otherwise normal haematological parameters, consistent with hereditary persistence of fetal haemoglobin (HPFH). An additional 16 cases labelled "raised HbF/HbF variant" included both HPFH and δβ-thalassemia carriers. Distinguishing HPFH from δβ-thalassemia has clinical relevance: HPFH is benign, while δβ-thalassemia in compound heterozygosity with β-thalassemia or HbS may cause moderate disease. HPLC alone cannot reliably distinguish these entities; gap-PCR or MLPA for delta-beta globin gene deletions is recommended.22

 

Rare Variants: HbE, HbD, HbQ, HbC, and Hb Lepore

Rare variants—including HbE trait (2 cases), HbD-Punjab trait (2 cases), HbQ trait (1 case), HbC (1 case), and Hb Lepore (1 case)—collectively constituted 7 cases (3.04% of abnormal). These findings demonstrate the value of a high-sensitivity HPLC-based screening programme in a diverse population. HbE is the most prevalent haemoglobin variant globally after HbS and is highly prevalent in north-east India and South-East Asia.23 HbE in compound heterozygosity with β-thalassemia (βE-thalassemia) is the most common form of severe thalassaemia in South and South-East Asia, reinforcing the importance of HbE carrier detection.

 

HbQ-alpha is a rare structural variant (alpha-globin chain substitution) that elutes in the A2 window on D10 chromatograms and may raise the apparent HbA₂. Failure to recognise HbQ may falsely diagnose β-thalassemia trait. In our case, peripheral smear showed essentially normal morphology, and the "elevated HbA₂" was on review recognised as a distinct HbQ peak.24

 

Hb Lepore (one case) presented as a P3-window peak with mild thalassaemic indices. Hb Lepore arises from non-homologous crossover between the δ- and β-globin genes, producing a δβ-fusion chain. It is clinically and haematologically similar to β-thalassemia trait in the heterozygous state and may cause severe disease when homozygous or compound heterozygous with β-thalassemia. Confirmation by DNA sequencing was essential in this case.25

 

Unknown Peaks

Unknown peaks (39 cases, 3.62%) represented a significant diagnostic challenge. These included samples with variant peaks at unusual retention times not identifiable by the D10 software. Cases were referred to higher centres for advanced characterisation. This highlights a recognised limitation of HPLC: uncommon or novel variants generating atypical retention-time patterns require supplementary techniques, including DNA sequencing, for definitive diagnosis.14,26

 

Role of HPLC in Paediatric Screening

HPLC remains the most widely recommended first-line tool for neonatal and paediatric hemoglobinopathy screening globally and in Indian guidelines.27,28 Its advantages include: simultaneous quantification of HbA, HbA₂, HbF, and structural variants in a single run; high throughput (~200 samples/8-hour shift on D10); excellent reproducibility (CV <1% for major fractions); and the ability to detect rare variants. In resource-constrained settings, HPLC combined with a targeted peripheral smear and sickling test provides a highly cost-effective diagnostic algorithm.

 

The high prevalence of β-thalassemia trait (10.77%) and sickle cell trait (1.58%) in our paediatric cohort has direct public health implications. Universal neonatal screening would allow early prophylactic penicillin and pneumococcal vaccine administration in SCA patients, significantly reducing infectious morbidity; early identification of β-thalassemia major enables timely enrolment in transfusion and chelation programmes; and carrier detection enables genetic counselling before reproduction.29,30

Strengths and Limitations

Strengths of this study include the large paediatric sample size (n=1077), the comprehensive multimodal diagnostic workflow (CBC, peripheral smear, sickling test, HPLC, and selective molecular analysis), and the identification of rare variants including HbQ, Hb Lepore, HbS+HbD compound heterozygotes, and the c.92+5G>C β-thalassemia mutation.

 

Limitations include: (i) the retrospective design with incomplete clinical follow-up data; (ii) non-uniform availability of iron studies, which may have caused underestimation of β-thalassemia trait in iron-deficient subjects; (iii) single-centre data that may not reflect the full epidemiological diversity of the region; and (iv) molecular analysis was performed in only a subset of cases, leaving some unknown peaks uncharacterised.

β-Thalassemia trait is the most common hemoglobinopathy in the paediatric population served by this tertiary centre, followed by sickle cell-related disorders and raised HbF variants. HPLC using the Bio-Rad D10 system is a sensitive and reliable first-line tool capable of identifying both common and rare haemoglobin variants, including HbQ, Hb Lepore, and HbS+HbD compound heterozygotes. Peripheral smear examination and the sodium metabisulphite sickling test remain indispensable adjuncts for resolving HPLC ambiguities. Molecular analysis is essential for unknown peaks, borderline cases, and confirmation of severe mutations such as c.92+5G>C in the HBB gene. The significant carrier burden identified in this study underscores the urgent need for structured neonatal and antenatal screening programmes, systematic genetic counselling, and comprehensive family studies to curtail the incidence of severe hemoglobinopathies in future generations.

 

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