Diagnosis of hemoglobinopathies: electrophoresis vs. HPLC

Alkaline cellulose acetate and acidic citrate agar electrophoreses are the most widely utilized methods for hemoglobin analysis. However, due to their limited resolution, incorrect or unresolved diagnosis of common hemoglobinopathies are sometimes encountered.Isoelectric focusing provides excellent resolution but is labor intensive and lacks accurate quantitation. High-performance liquid chromatographic methods have been developed for either screening or confirmation of hemoglobinopathies with relatively high sensitivity or specificity. Through the years, we have developed, refined and optimized an HPLC procedure using a porous silica coated with polyaspartic acid to improve the elution time of hemoglobin analysis while maintaining the high sensitivity and resolution necessary for both screening and confirmatory purposes.The method is capable of separating more than 45 commonly encountered hemoglobin variants within 12 min. These include Barts, H, A1C, Raleigh, Hope, I, F, Camden, N-Baltimore, I-High Wycombe, I-Paris, J-Baltimore, N-Seattle, Grade, Fannin-Lubbock, Malmo, South Florida, A, Chicago, G-Georgia, Lepore-Baltimore, P-Galveston, G-Coushatta, Lepore-Boston, E, Zurich, Osu Christiansborg, A2, G-Philadelphia, Korle Bu, Russ, E-Saskatoon, Richmond, D-Punjab, Deer Lodge, Koln, Montgomery, S, Q-Thailand, G-San Jose, A2', Hasharon, Q-India, Tampa, Constant Spring, SG-hybrid, C-Harlem, O-Arab, British Columbia, and C. The method provides not only the identification of the aforementioned hemoglobin and variants but also an accurate quantitation of their concentrations, particularly Hb F and A2, which are useful for the diagnosis of HPFH and beta-thalassemia, respectively.The simplicity of the sample preparation, superior resolution of the method, and accurate quantitation of hemoglobin concentration, combined with complete automation, make this an ideal methodology for the routine diagnosis of hemoglobin disorders in a clinical laboratory.