Quantitation of Three Types of γ Chain of HbF by High Pressure Liquid Chromatography; Application of This Method to the HbF of Patients With Sickle Cell Anemia or the S-HPFH Condition

A modification of a high pressure liquid chromatographic (HPLC) procedure19 is described that enables the complete separation and quantitation of the AγT, AγI and Gγ chains in human fetal hemoglobin. The method, which is fast and accurate, requires 5 to 2000 µg Hb F. The purity of the Hb F is not essential and admixture of up to 70% adult Hb does not interfere with the determination. The method has been applied to the Hb F of 64 Black SS patients and 7 persons with the Hb S-HPFH (GγAγ type) condition. (A) Both “adult” Gγ to Aγ (2:3) and “newborn” Gγ to Aγ (3:2) ratios were observed in adult SS patients, 8 yr and older. Only 12% of the SS patients had the “newborn” ratio. This high Gγ to Aγ ratio may be due to a modification of the genetic switch mechanism that regulates the change of this ratio after birth.8 (B) Intermediate Gγ to Aγ ratios were only found in young SS patients, 5 yr of age or less. The results suggest a delayed switch of the newborn adult ratio in sickle cell anemia. (C) The AγT chain was present in only 6% of all SS patients.12 One patient is homozygous for this variant chain. (D) Three of the 7 subjects with Hb S-HPFH were positive for the AγT chain. Its percentage was low, which suggests that the AγT chain gene is in trans of the HPFH determinant. (E) Quantitation of the three γ chain types is also possible in the Hb F from Hb S heterozygotes with (nearly) normal Hb F levels. Such an analysis is useful for an evaluation of genetic conditions involving variations in the production of (different types of) Hb F.