Utility of First-Line Diagnostic Tests and Molecular Methods for Accurate Characterization of Sickle Cell Disorders

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.

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.

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 (HbSD^Punjab) 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.

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.