Simplified hemolysate preparation for identification and quantitation of hemoglobin by electrophoresis

Abstract

A simplified hemolysate-preparation reagent was composed of 1 g of ethylenediamine tetraacetic acid and 0.1 g of saponin dissolved in 500 ml of distilled water. The hemolysate was prepared by adding equal volumes of the reagent into normal-saline-washed packed red cells and mixing for five minutes. The hemolysates of 109 samples included 24 normal individuals, 21 beta-thalassemia trait, 23 heterozygotes of hemoglobin E, 21 hemoglobin H disease and 20 cord blood samples with hemoglobin Bart's, which were tested by cellulose acetate electrophoresis to identify hemoglobin types and their concentration. The results were compared with those obtained from standard preparation method using a paired-t-test. There was no significant difference of the hemoglobin concentration from the two preparation methods (p = 0.1718). There was good correlations between the two methods of hemolysate preparation with a correlation coefficient of 0.9887 and a regression line of Y=0.9678X+0.1633. The cellulose acetate electrophoresis of simplified hemolysate showed clear separation of each hemoglobin bands. We concluded that the simplified hemolysate preparation could be used in the laboratory for identification and quantification of hemoglobin. The advantages of this hemolysate preparation method are it is simple to perform and contact with chemical hazard can be avoided.

Keywords

• hemolysate preparation
• hemoglobin electrophoresis
• thalassemia
• toluene