Usefulness of Cation Exchange High Performance Liquid Chromatography as a Testing Procedure

Abstract

Testing of cord blood or newborn blood samples for hemoglobin abnormalities should include clinically important hemoglobinopathies other than sickle cell anemia (SS), such as SC, SD, SO, S-β- thalassemia (thal), EE, SE, and α-thal, and should place the quality of the testing procedures (ie, accuracy of diagnosis) above quantity (ie, number of samples tested over a given period). There is no single method available that is suitable for the identification of each of the numerous abnormalities; thus, at least two, and often more than two, procedures must be used to reach a definitive diagnosis. For this reason, blood samples collected in vacutainers with ethylenediaminetetraacetic acid as anticoagulant are preferred to those collected on filter papers. The latter approach also has the disadvantage that, under a less than optimal transport system, hemoglobin is readily modified (oxidation, glycosylation, protein-protein interaction), producting extra bands or peaks in electrophoretic or chromatographic separations that interfere with an appropriate identification of various genetically determined hemoglobin variants. In our laboratories, in which hemoglobin identification has been routine for more than 25 years, we consider the following procedures acceptable primary testing methods: starch gel electrophoresis at pH 8.9, cellulose acetate electrophoresis at pH 8.5 to 8.9, isoelectric focusing, and fast cation exchange high performance liquid chromatography (HPLC). The following five methods are excellent confirmatory testing procedures: citrate agar electrophoresis at pH 6.1, cation or anion exchange macrochromatography, isoelectric focusing, cation exchange HPLC, and immunologic procedures. Combinations of these techniques will often lead to acceptable data, and the general approach followed in our institute is given in Fig 1. Cellulose acetate electrophoresis at alkaline pH is still the primary testing procedure, and citrate agar electrophoresis at pH 6.1 and micro-HPLC procedures are the main confirmatory methods.