Abstract
Hemoglobin High-performance liquid chromatography (Hb HPLC) is a standard first-line technique for diagnosis of thalassemia and hemoglobinopathies. We compared two HPLC systems for detection and quantification of normal and abnormal Hb fractions. EDTA samples from 100 normal healthy subjects and 107 subjects affected with hemoglobinopathies or carriers were analysed using HPLC systems Tosoh HLC-723G11 and Bio-Rad Variant-II. Retention time (RT) and area of peaks for HbA2, HbF and other structural variants were compared. In discrepant cases samples were run on Sebia Capillary zone electrophoresis (CZE) for confirmation of results (39 out of 107 cases with HbE, HbD Iran, Hb Lepore and HbQ). Measurement of HbA2 and HbF in normal samples and HbF in those with variant Hbs showed good correlation by both analyzers (R2 = 0.83, 0.9 and 0.99 respectively). HbE co-elutes with HbA2 in Bio-Rad. Correlation done using the apparent HbA2 concentration from Bio-Rad with (HbE + HbA2) from Tosoh G11 showed good correlation (R2 = 0.97). Correlation of HbS (Eluting at S-window at RT 3.11 min in Tosoh G11 and 4.33 min in Bio-Rad) as well as HbD Punjab (Eluting at D-window at RT 2.82 min in Tosoh G11 and 4.06 min in Bio-Rad) by both instruments was good. HbD Iran (Eluting at E-window at RT 2.69 min in Tosoh G11 and with HbA2 at 3.53 min in Bio-Rad); HbQ (Eluting at C-window at RT 3.78 min in Tosoh G11 and unknown window at 4.7 min in Bio-Rad), HbH (Eluting at P00 window at RT 0.13 min in Tosoh G11 and giving pre-integration peak in Bio-Rad), Hb Lepore (Eluting at P08 window at RT 2.67 min in Tosoh G11 and with HbA2 at 3.46 min in Bio-Rad) gave comparable results. Correlation with findings of CZE was done in few cases when needed. Two automated HPLC instruments demonstrated similar usefulness for screening patients for hemoglobinopathies. However, complex elution patterns as well as co-elution of variants like HbA2, HbE, Hb Lepore, HbD Iran (in Bio-Rad); HbD Iran and HbE (Tosoh G11) pose difficulty in interpretation. A complementary second method like CZE may be required.
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Supplementary Fig. 1: Chromatograms of Bio-Rad Variant II and Tosoh G11in cases of thalassemia trait (a, b) and thalassemia homozygous (c, d) (JPEG 164 kb)
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Supplementary Fig. 2: Chromatograms of Bio-Rad Variant II and Tosoh G11in cases of HbS trait (a, b), homozygous (c, d) and sickle beta compound heterozygous (e, f) (JPEG 274 kb)
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Supplementary Fig. 3: Chromatograms of Bio-Rad Variant II and Tosoh G11in cases of HD Punjab heterozygous (a, b), homozygous (c, d) and HbH (e, f) (JPEG 342 kb)
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Supplementary Fig. 4: Chromatograms of Bio-Rad Variant II, Tosoh G11 and Sebia CZE in cases of Hb Lepore (a–c), HbD Iran (d–f) and HbQ India (g–i) (JPEG 468 kb)
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Supplementary Fig. 5: Chromatograms of Bio-Rad Variant II, Tosoh G11 and Sebia CZE in normal control without any hemoglobin variants (a–c) (JPEG 130 kb)
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Chopra, P., Bhardwaj, S., Negi, P. et al. Comparison of Two High-Pressure Liquid Chromatography Instruments Bio-Rad Variant-II and Tosoh HLC-723G11 in the Evaluation of Hemoglobinopathies. Indian J Hematol Blood Transfus 36, 725–732 (2020). https://doi.org/10.1007/s12288-020-01298-5
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DOI: https://doi.org/10.1007/s12288-020-01298-5