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Laboratory methods allowing the detection and characterization of hemoglobin variants are reviewed. Protein chemistry techniques such as isoelectrofocusing, electrophoreses under various experimental conditions, cation exchange and reversed phase high performance liquid chromatography, are the most frequently used for the detection of variants. When associated with a few additional data they may lead to a presumptive diagnosis. DNA studies are also developed in many laboratories. Final identification of a variant may be achieved either by molecular biology techniques or by protein sequence analysis in which mass spectrometry now occupies a key position.

Hb Mont Saint-Aignan [beta128(H6)Ala-->Pro] is a mildly unstable variant, associated with hemolytic anemia, marked microcytosis and increased alpha/beta biosynthetic ratio (1.55 versus 1.1 +/- 0.1 in the control). The abnormal chain was isolated by selective precipitation with isopropanol and the structural modification determined by protein chemistry methods (reversed phase high performance liquid chromatography and mass spectrometry). Possible mechanisms underlying the beta(+)-thalassemia-like expression of this variant are discussed.

We here report two new, clinically silent, hemoglobin variants in which the structural modification disturbs the oxygen-linked chloride binding. Hb Antananarivo [alpha1(NA1)Val-->Gly] was found during a systematic hematological study in a 24-year-old woman, who originates from Madagascar. Hb Barbizon [beta144 (HC1)Lys-Met] was found in several members of a French family. The oxygen binding properties of Hb Barbizon were similar to those of Hb Antananarivo showing, in vitro, a decreased chloride effect as compared to Hb A. In Hb Barbizon, the replacement of lysine beta144 by a methionine residue decreased from 4 to 2 the excess positive charges in the central cavity, thus leading to a reduction of about half of the chloride effect. For Hb Antananarivo, the mechanism is unclear since there is no difference in the number of positive charges in the central cavity but alterations are likely at the alpha1alpha2 interface.

The two classical methods used to determine the level of Hb F in a hemolysate are herein described. Measurement of this hemoglobin fraction by resistance to alkali denaturation was the first technique introduced; it is today largely replaced by ion exchange high performance liquid chromatography. The limits, pitfalls, and advantages of the two methods are discussed.