Simplified hemoglobin chain detection by capillary electrophoresis.
Hemoglobin (Hb) chains have been analyzed traditionally by cellulose acetate electrophoresis after sample extraction with acetone and denaturation with concentrated urea in order to detect thalassemia (Thal). A few capillary electrophoresis (CE) methods have been also described for separation of Hb chains also after sample extraction. We describe a CE method for analysis of Hb chains without sample preparation. Red blood cells were diluted (hemolyzed) in water and injected directly onto the capillary. The separation was performed in concentrated phosphate buffer at pH 12.6 and 2.15. Under these conditions of pH and buffer concentration, the chains were denatured and separated from the heme during electrophoresis. The common variants of the beta-chains, such as beta(S), beta(C), and beta(E), are also separated from each other. The intact Hb molecule is analyzed using the same sample and CE conditions but in an arginine-Tris buffer, pH 8.6. The data from the three separations are used to complement each other for interpretation of the presence of Hb variants and for thalassemia. The main advantages of this method are simplicity and speed. This method illustrates the flexibility and simplicity of the CE for analysis of the hemoglobinopathies.
Shihabi
Zak K
ZK
Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA. zshihabi@wfubmc.edu
Hinsdale
Mark E
ME
D002021:Buffers; D019075:Electrophoresis, Capillary / Q000379:methods*; D005914:Globins / Q000302:isolation & purification*; D006453:Hemoglobinopathies / Q000175:diagnosis; D006454:Hemoglobins / Q000032:analysis*; D006455:Hemoglobins, Abnormal / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013789:Thalassemia / Q000097:blood* / Q000175:diagnosis
D002021:Buffers; D006454:Hemoglobins; D006455:Hemoglobins, Abnormal; D005914:Globins