title: Qualitative and quantitative analysis of hemoglobin variants by capillary isoelectric focusing.
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.
authors.affiliation: Service de Biochimie A, Hôpital Saint-Antoine, Paris, France.
authors.forename: N
authors.initials: N
authors.lastname: Mario
authors.forename: B
authors.initials: B
authors.lastname: Baudin
authors.forename: J
authors.initials: J
authors.lastname: Giboudeau
mesh_terms: D004563:Electrochemistry; D005319:Fetal Hemoglobin / Q000032:analysis / Q000302:isolation & purification; D006442:Glycated Hemoglobin / Q000032:analysis / Q000302:isolation & purification; D006441:Hemoglobin A / Q000032:analysis / Q000302:isolation & purification; D006451:Hemoglobin, Sickle / Q000032:analysis / Q000302:isolation & purification; D006454:Hemoglobins / Q000032:analysis* / Q000737:chemistry; D006455:Hemoglobins, Abnormal / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D009995:Osmosis
chemical_list: D005319:Fetal Hemoglobin; D006442:Glycated Hemoglobin; D006441:Hemoglobin A; D006451:Hemoglobin, Sickle; D006454:Hemoglobins; D006455:Hemoglobins, Abnormal