Abstract
The authors describe a modification of the instrumental parameters of the Diamat fully automated HPLC system for Hb A2 assay (Bio-Rad Laboratories, Milan, Italy) in order to obtain simultaneous determination of Hb A2 and Hb F.
Hb A2 and Hb F measurements are reproducible (within-run CV 2.6%, with Hb A22.7%; 5.1%, with Hb F 1.3%) and accurate (from a comparison with two microchromatographic techniques for Hb A2: r = 0.9639 and 0.9755; with two alkali denaturation procedures for Hb F: r = 0.9990 and 0.9952; with radial immunodiffusion, r = 0.9877). Assay linearity has been confirmed for Hb A2 concentrations between 0 and 6.0%, and for Hb F between 0 and 60%. The data obtained from the analysis of some pathological samples for Hb Bart's, Hb H, Hb J Sardegna, Hb Lepore and Hb S are in agreement with cellulose acetate electrophoresis analysis.
The Hb A2 reference intervals for normals (N = 597) and Beta-thalassemia carriers (N = 200) are respectively (95% limits) 2.02-3.27 and 3.92-5.90 in % units. Hb F values measured in normals (N = 968), in β-thal carriers (N = 302) and in δβ-thal carriers (N =3) have been found to be consistent with the usual diagnostic parameters.
Some minor limitations emerged: the most relevant concerns Hb A1c, which is overestimated with respect to a reference method (y = 1.217x + 0.16; N = 79; r = 0.9235). A probable interference from labile fractions is responsible for this Hb A1c inaccuracy.
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