Variant 2 β thalassemia short program

Hb variants were detected by HPLC. The VARIANT™ II β-thalassemia Short Program (Bio-Rad, Hercules, CA, USA) method, which separates Hb variants by cation-exchange chromatography using a salt gradient, was used, with the calibrators and controls provided by the manufacturer with every batch. The analysis consisted of monitoring retention times, area percentages, and concentrations of various peaks and windows for different Hb variants: HbF (retention time of 1.1 min with 0.98–1.2 min window), HbA0 (2.5 min and 2.0–3.0 min), HbA2 (3.65 min and 3.57–3.75 min), and minor peaks, such as P2 (1.39 min and 1.28–1.5 min) and P3 (1.7 min and 1.5–1.9 min).

The remaining erythroid cells on day 14 were all collected and washed once with 1X DPBS. The cell pellet was lyzed for hemoglobin measurement by HPLC using the VARIANT^™ II β-thalassemia Short Program (Biorad, Hercules, CA, USA). This cation exchange HPLC technique uses an increasing sodium phosphate gradient buffer to separate different hemoglobin variants. Hemoglobin was measured using absorbance at 415 nm, and correction for turbidity was done by the absorbance at 690 nm [21 (link)]. Each 6.5-minute assay detects the most prevalent aberrant hemoglobin variations while also providing quantitative findings for the percentages of HbA2/E and HbF.