Electrophoretic and chromatographic techniques for the differential diagnosis of a haemoglobin abnormality: Hb E heterozygosity

A method is described for separating haemoglobin (Hb) E (beta 26 Gly----Lys) from Hb A2 (a normal minor Hb component in adult blood). The technique allows the distinction between subjects carrying beta-thalassaemia trait and patients who are simultaneously alpha-thalassaemic and heterozygous for Hb E, the standard electrophoretic pattern often being similar in these two circumstances. Complete separation between Hb E and Hb A2 (2 mm empty space in between) is obtained by isoelectric focusing in immobilized pH gradients in an ultra-narrow pH 7.55-7.65 gradient. The apparent pI values of the two species (at 10 degrees C and at an average ionic strength of 5.6 mequiv. l-1) have been calculated to be 7.603 for Hb E and 7.607 for Hb A2. Thus, the system reported here affords a resolution of at least 0.004 pH unit.