A comprehensive analysis of hemoglobin variants by high-performance liquid chromatography (HPLC)

Introduction: High-performance liquid chromatography (HPLC) is a method commonly used for the detection of hemoglobin (Hb) variants. In addition to providing precise quantitation of Hb A(2) and Hb F, the reported retention time and peak shape of a high number of hemoglobin (Hb) variants are very helpful for presumptive identification. However, there is a scarcity of summarized data in the literature of the mobility of Hb variants on this method. Methods: A total of 383 Hb variants were studied on the Bio-Rad Variant (TM) Classic HPLC instrument. Hb variant identification used a number of methods, including confirmation by DNA sequencing in at least one case for all alpha and beta chain Hb variants. Results: Retention time data and the number of occurrences of each Hb variant were obtained. This showed that rare Hb variants can have similar retention times to the five most common alpha or beta chain Hb variants. Conclusion: HPLC is a very powerful tool in the evaluation of Hb variants, particularly when combined with other methods. However, it should not be used as a stand-alone method for definitive identification of Hb variants.