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Updated: Mar 3, 2026
A Precision Medicine Tool for Measurement and Monitoring of Hemoglobin S in Sickle Cell Disease Patients Receiving Transfusion Therapy
Utility of First-Line Diagnostic Tests and Molecular Methods for Accurate Characterization of Sickle Cell Disorders
Diksha Dev Yadav1, Sanjeev Chhabra1, Jasbir Kaur Hira1
1Department of Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Insights
Sickle cell disorder diagnosis relies on HPLC and hemogram data, with sickling tests and molecular analysis for complex cases. Accurate classification is crucial for effective patient management and understanding disease variants.
Area of Science:
- Hematology
- Clinical Diagnostics
- Genetic Disorders
Background:
- Sickle cell disorders (SCD) are prevalent hemoglobinopathies globally.
- Accurate diagnosis requires multiple tests like complete blood count, HPLC, electrophoresis, sickling tests, and molecular assays.
- Evaluating diagnostic utilities of these methods is essential for precise SCD classification.
Purpose of the Study:
- To assess the diagnostic performance of various testing methods for precise classification of SCDs.
- To compare the effectiveness of HPLC, electrophoresis, sickling tests, and molecular assays in a specialist hematology setting.
Main Methods:
- Analysis of clinical, demographic, and laboratory data for SCD cases diagnosed between 2009 and 2020.
- Utilized CE-HPLC, slide-based sickling test, and cellulose acetate electrophoresis.
- Conducted molecular testing for HbS and β-thalassemia mutations in select cases.
Main Results:
- Identified 261 HbS variant cases: 56% sickle cell trait, 21% Sβ-thalassemia, 17% sickle cell anemia, and 5% HbSD-Punjab.
- HbA2 ≥ 4.0% showed high sensitivity (95.2%) and specificity (96.5%) in differentiating SCA from Sβ-thalassemia.
- Alkaline pH electrophoresis and sickling tests demonstrated 100% sensitivity; PCR-RFLP had a diagnostic pitfall.
Conclusions:
- HPLC and hemogram data reliably characterize most SCDs in hospital settings.
- Sickling tests and parental HPLCs supplement diagnosis when needed.
- Molecular analysis is valuable for complex cases but has potential pitfalls.
Introduction:
Sickle cell disorders (SCD) are among the commonest symptomatic hemoglobinopathies worldwide. Their diagnosis involves multiple tests, including complete blood count, cation-exchange high-pressure liquid chromatography (CE-HPLC), hemoglobin electrophoresis, sickling tests, and molecular assays. We evaluated the diagnostic utilities of various testing methods for precise classification of SCDs in a specialist hematology laboratory of a tertiary-care teaching hospital.
Methods:
Clinical, demographic and laboratory data from all SCDs diagnosed between January 2009 and June 2020 using CE-HPLC, slide-based sickling test and cellulose acetate electrophoresis were analyzed. Cases with over 50% S-window peaks on CE-HPLC were considered for HbS and β-thalassemia mutation testing. Performance indices of CE-HPLC, sickling test and electrophoresis were determined in molecularly-proven SCDs.
Results:
The 261 cases with HbS variant included 56% (n = 147) sickle cell traits (SCT), 21% (n = 55) Sβ-thalassemia (Sβ), 17% (n = 45) sickle cell anemia (SCA), and 5% (n = 14) HbSD-Punjab (HbSDPunjab) cases. Molecular analysis of 71 cases revealed that HbA2 ≥ 4.0% discriminated most efficiently between SCA and Sβ, with 95.2% sensitivity, 96.5% specificity and positive/negative predictive values of 97.5%/93.3%. The commonest β-thalassemia mutation in Sβ-thalassemia was HBB:c.92 + 5G>C (58.4%). Alkaline pH electrophoresis and sickling test showed 100% sensitivity. A diagnostic pitfall occurred in PCR-RFLP due to loss of DdeI restriction site.
Conclusions:
Most SCDs encountered in a hospital-setting are reliably characterized by HPLC and hemogram data, supplemented with the sickling test and, where necessary, parental HPLCs. Molecular analysis aids in accurate characterization of complex cases and in resolution of diagnostic discrepancies; however, their own pitfalls must be borne in mind.
