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  • Title: Qualitative and quantitative analysis of hemoglobin variants by capillary isoelectric focusing.
    Author: Mario N, Baudin B, Giboudeau J.
    Journal: J Chromatogr B Biomed Sci Appl; 1998 Feb 27; 706(1):123-9. PubMed ID: 9544814.
    Abstract:
    We developed two capillary isoelectric focusing (CIEF) assays, in narrow pH gradients, with the aim of routinely separating and quantitating normal and abnormal hemoglobins (Hbs): a one-step CIEF assay where residual electroosmotic flow mobilizes the proteins during focalization, and a two-step CIEF assay where focused Hbs are mobilized by low pressure by maintaining high-voltage. The resolution of 0.10 pH unit obtained with the one-step assay allowed the separation of the Hbs A, F, S and C; but Hb A2, which represents about 2-3% of whole Hb, could not be quantitated. The better resolution of 0.02 pH unit obtained with the two-step assay allowed the separation of some Hb variants of very close isoelectric points. The reproducibility of retention times was satisfactory (C.V.<5%). Moreover, in this configuration quantitation of Hb A2, Hb F and Hb S led to a standard deviation of less than 5%, allowing the diagnosis of thalassemias. The one-step assay could be useful only for the detection of abnormal variants, while the two-step assay could be applied to the routine analysis of Hbs, with quantitation of minor fractions and presumptive identification of variants.
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