Comparison of Capillary Zone Electrophoresis with High-pressure Liquid Chromatography in the Evaluation of Hemoglobinopathies

Abstract

Objective: The capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC) methods were compared in terms of HbA2 measurement for the assessment of hemoglobinopathies.

Materials and methods: CZE was compared with HPLC for the evaluation of patients without hemoglobinopathy (n=321), with β-thalassemia trait (n=113), and with common (HbD-Punjab, E, C, S/A, and S/S) and rare (HbS/D, O-Arap, Lepore, G-Coushata, Setif, Hamadan, Q-Iran, and H) variants (n=21). The reference range for HbA2 was determined by CZE.

Results: Among patients without hemoglobinopathy, the median (2.5^th-97.5^th percentiles) values were 97.4% (97.0-98.0%) and 97.5% (96.6-98.4%) for HbA (p=0.060) and 2.4% (1.6-3.0%) and 2.5% (1.6-3.1%) for HbA2 (p<0.001) by HPLC and CZE, respectively. The reference range for HbA2 was 1.6-3.1% by CZE. In the comparison of methods for HbA2, there was a constant error of 0.255 (confidence interval: 0.062-0.448) and bias of 0.10% (limit of agreement: 0.33-0.53), and higher values were obtained with CZE. A strong correlation was observed between the methods (r=0.782). Interrater agreement was almost perfect for clinical diagnosis (ϰ=0.911). The two methods detected and identified the common variants similarly. All rare variants, except HbH by HPLC and HbS/D by CZE, were detected as separate peaks by both methods.

Conclusion: The two methods were in agreement regarding the preliminary identification of β-thalassemia patients. Different Hb variants were detected by both methods but with possible methodological interference for HbA2 measurements. CZE is a reliable and simple alternative for the evaluation of hemoglobinopathies. The standardization of HbA2 measurements should be prioritized as more techniques become available in routine laboratory practice.

Keywords: Anemia; Thalassemia; Other hemoglobinopathies; Hemoglobin disorders.

Figure 1

Deming regression and Bland-Altman analyses for HbA2 at 209x297 mm (300x300 DPI).

Figure 2

Comparison of HPLC and CZE reports for the rare Hb variants. A) Hb H: Migrated at zone 15 with CZE but was not detected by HPLC. B) Hb S/D: Hb D (39.6%) and HbS (33.3%) were detected with HPLC while 77.2% Hb variant was detected in the D zone with CZE. C) Hb-O-Arap: Migrated at zone A2 with CZE.

Figure 3

HPLC, CZE, and DNA analyses for the rare α-chain Hb variants. A) Hb Setif: p.Asp95Tyr (c.283G>T); 14.5% and 15.1% variant Hb was detected by HPLC and CZE, respectively. B) HbQ-Iran: p.Asp76His (c.226G>C); 19.4% and 19.3% variant Hb was detected by HPLC and CZE, respectively.