Detection of hemoglobinopathies by high-performance liquid chromatography in patients with microcytic hypochromic anemia in Western Nepal

Study design and setting

A hospital-based cross-sectional study was conducted at Nepalgunj Medical College Teaching Hospital, Banke, Nepal, from October 2023 to September 2024.

Sample size and sampling

Sample size was calculated using n = (Z² × p × q)/d², where Z = 1.96, p = 0.158,^[14] q = 0.842, and d = 0.05, yielding 205 patients. After 10% adjustment, the final sample size was 228. Consecutive sampling recruited patients with microcytic hypochromic anemia.

Inclusion and exclusion criteria

Data collection

Five milliliters of venous blood were collected in ethylenediaminetetraacetic acid tubes. Complete blood count was analyzed using the Sysmex XN-1000 automated hematology analyzer within 2 h. Hemoglobin variant analysis used the Bio-Rad D-10 system. Quality control included daily calibration with manufacturer-supplied calibrators; internal quality control with normal and abnormal controls run and participation in external quality assessment programs coordinated by regional reference laboratories. Inter-assay and intra-assay coefficients of variation remained below 3%.

HPLC interpretation criteria

Hemoglobin fractions were interpreted using established thresholds:^[15] β-thalassemia trait (hemoglobin A2 >3.5%), sickle cell trait (hemoglobin S 20–50%), sickle cell disease (hemoglobin S ≥50%), hereditary persistence of HbF (HbF >10% in absence of other causes with characteristic HPLC pattern showing predominant HbF without elevated HbA2).

Statistical analysis

Data were analyzed using the Statistical Package for the Social Sciences version 26. Descriptive statistics included frequencies, percentages, mean, and standard deviation. Independent t-test compared continuous variables between the β-thalassemia trait and non-thalassemia groups. Given the descriptive objectives of this study, multivariate analysis was not performed. Statistical significance was P < 0.05.

Ethical considerations

Institutional Review Committee approval was obtained (Ref: 73/081-082). Written informed consent was obtained from adult participants or legal guardians for pediatric participants (<18 years). Participants with abnormal results received counseling, written reports, and appropriate referrals.

Study population characteristics

A total of 228 patients were enrolled. Mean age was 30.96 ± 17.28 years (range: 0.66–85 years) with female predominance (75.9%). MCV of 70.54 ± 7.36 fL confirmed universal microcytosis. Table 1 summarizes demographic and hematological characteristics.

Prevalence of hemoglobinopathies

Hemoglobinopathies were detected in 45 patients (19.7%). β-thalassemia trait was most common (10.1%), followed by sickle cell trait (7.5%), sickle cell disease (1.3%), and hereditary persistence of HbF (0.9%) as shown in Table 2.

Distribution of hemoglobin variants

Among β-thalassemia trait cases, hemoglobin A2 ranged from 3.6 to 6.8%. Five cases (21.7%) showed borderline values (3.5–3.9%). Studies from similar populations suggest that most individuals with borderline HbA2 levels harbor β-globin mutations, though confirmatory molecular testing was not performed in our study. Concurrent iron deficiency, which can suppress HbA2 levels, may also contribute to borderline values. Sickle cell trait cases showed hemoglobin S 20–45% (mean 35%). Three sickle cell disease cases demonstrated hemoglobin S 62–71% with elevated HbF (8–15%).

Comparison of hematological parameters

Mean hemoglobin A2 was significantly higher in β-thalassemia trait (4.95 ± 0.66%) versus non-thalassemia cases (2.38 ± 0.44%, P < 0.001). HbF, MCV, and hemoglobin showed no significant differences [Table 3].

Prevalence and clinical spectrum

Our 19.7% prevalence in this selected hospital population aligns with regional studies.^[4,6,16,17] The 10.1% β-thalassemia trait prevalence is comparable to other South Asian regions.^[18] Detection of sickle cell variants (8.8% combined) is noteworthy, as these disorders have been historically underrecognized in Nepal. The Terai region’s ethnic diversity, including populations with ancestral links to tribal communities, may explain this finding.^[10]

Diagnostic importance of MCV, red cell distribution width (RDW), and HPLC

Our findings demonstrate that MCV alone cannot differentiate between IDA and β-thalassemia trait, as both conditions produce a microcytic blood picture. While MCV combined with RDW can provide better diagnostic clues with thalassemia trait, typically showing normal RDW and IDA showing elevated RDW, these indices remain insufficient for definitive diagnosis.

The diagnostic complexity is further increased by frequently coexisting conditions. β-thalassemia trait with concurrent iron deficiency is particularly common in our population, and β-thalassemia trait with vitamin B12 deficiency, though less common, also complicates the blood picture. In such cases, the characteristic hemoglobin pattern may be masked, making clinical diagnosis based on indices alone unreliable.

This shows why HPLC is of paramount importance in detecting hemoglobinopathies in the community. Our study reveals that conventional hematological parameters completely failed to distinguish β-thalassemia trait from other causes of microcytic anemia. MCV (P = 0.757), hemoglobin level (P = 0.825), and HbF (P = 0.293) showed no significant differences between groups. Only hemoglobin A2 quantification by HPLC provided definitive discrimination (P < 0.001). This establishes HPLC not as an optional refinement but as the mandatory diagnostic method for accurately classifying microcytic anemia in high-prevalence populations.

Regional considerations for HbA2 cutoff values

The selection of appropriate HbA2 cutoff values remains crucial for accurate diagnosis. While we employed the internationally accepted 3.5% threshold, regional variations exist with cutoffs ranging from 3.3% to 4.0%.^[19,20] Mohanty et al. used HbA2 >4.0% in tribal populations in Rajasthan, India, reporting 7.25% β-thalassemia trait prevalence.^[7,8,21] Similarly, a large multicentric Indian study used HbA2 >4.0% as the diagnostic cutoff.^[4,6,22,23] Had we adopted the 4.0% cutoff, 5 of our 23 β-thalassemia trait cases (21.7%) with borderline HbA2 values (3.5–3.9%) would have been misclassified as normal, reducing prevalence from 10.1% to 7.9%.

The clinical significance of borderline HbA2 cases cannot be understated. Studies show that 82% of individuals with borderline HbA2 levels (3.0–3.9%) harbor molecular or acquired defects with 73% demonstrating confirmed β-thalassemia mutations, typically β⁺ or β⁺⁺ variants producing milder phenotypes.^[19,20,24] When such “borderline normal” individuals partner with another carrier, offspring remain at 25% risk for β-thalassemia major. Several laboratories have revised cutoffs to 3.5% or 3.2% for diagnosis, as borderline cases are common in high-prevalence populations.^[12,25]

Given Nepal’s ethnic proximity to northern India, where similar β-globin mutation profiles exist, and the documented presence of hemoglobinopathies in our region, our use of the 3.5% cutoff appears appropriate for maximum sensitivity.^[5,10] The substantial proportion (21.7%) of borderline cases underscores the importance of comprehensive evaluation including iron studies, family screening, and when feasible, molecular confirmation for cases in the 3.5–3.9% range.

Comparison with previous studies

Our prevalence falls within South Asian ranges. Philip et al. found 15.8%, while Indian centers reported 15–30%.^[8,14,23] Shrestha et al. reported 12% in eastern Nepal.^[6,9,26] Our study provides crucial baseline data for this geographically distinct population presenting with microcytic anemia. The Bio-Rad HPLC system demonstrated excellent performance with >95% concordance with molecular testing for common variants in validation studies.^[15,26]

Public health implications and recommendations

The 19.7% prevalence in this hospital-based microcytic anemia population suggests that HPLC-based screening warrants consideration as part of routine clinical practice in high-prevalence regions. Such screening could prevent inappropriate iron therapy in a substantial proportion of microcytic anemia patients and enable informed genetic counselling for reproductive-age individuals. Proposed strategies for consideration include implementing HPLC screening for that population, establishing partner screening programs for identified carriers, and developing provider training programs emphasizing the distinction between genetic and nutritional causes of microcytosis as well as prioritizing HPLC installation in district hospitals serving high-prevalence regions, and developing a national hemoglobinopathy registry. These interventions may be economically justified when prevalence exceeds 5%.^[27]