Abstract
A rapid and inexpensive method is described for the enrichment of fetal hemoglobin (HbF) which eliminates the interference of other hemoglobins in the HPLC analysis of γ chains when HbF is ⩽ 20–30% of the total Hb. The enrichment procedure, which is carried out on 1 ml hemolysate, is based on the alkaline denaturation of the other Hbs followed by Zn 2+ precipitation. Samples are injected into the HPLC apparatus without further treatment. The method was validated by HPLC analysis of hemolysates with high levels of HbF and mixtures prepared by diluting the high HbF hemolysates with adult hemolysates. The relative proportions of γ chains as well as their chromatographic behavior were unaltered by the HbF enrichment procedure. The method is illustrated by the analysis of hemolysates of normal newborns and of patients with thalassemia, sickle cell diseases and aplastic anemia containing 3.4 to 100% HbF. For the four hemolysates containing > 20% HbF, the same quantitative and chromatographic results were obtained by direct analysis and after enrichment. Although reproducible and accurate results were obtained for the enrichment method and HPLC analysis when HbF was ⩾ 3%, at lower concentrations the variability of both was unacceptably high, indicating the need for additional or improved methodology for hemolysates containing very low levels of HbF.
Published Version
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