Purified Haemoglobin Preparations in the Evaluation of HbA 1c Determination by Ion Exchange Chromatography

Abstract

The use of ion exchange resins for the estimation of HbA_1c in clinical samples rests on the assumption that HbA_1c is effectively and efficiently separated from other N-terminally modified haemoglobins and from HbA_o. To test this assumption, we applied highly purified preparations of HbA_1a+1b, HbA_1c and HbA_o to ion exchange minicolumns, using conditions of application simulating actual blood samples and the first and second elution buffers provided by the manufacturer. The authenticity and purity of the applied haemoglobin preparations were documented by high performance liquid chromatography, gel electrophoresis and carbohydrate content. About 40% of the applied HbA_1a+1b eluted in the first fraction; 45% eluted in the second fraction, and 10% to 15% required 1 mol/L NaCl to elute from the column. Of the applied HbA_1c, 65–80% eluted where expected in the second fraction, about 20% required 1 mol/L NaCl to elute from the column, and the remainder eluted with HbA_1a+1b. Some 3–6% of pure HbA_o applied to minicolumns emerged in the second fraction, with the remainder eluting as expected after making the buffer 1 mol/L in NaCl. The results indicate that the fraction eluting from ion exchange minicolumn chromatography that is designated ‘HbA_1c’ contains HbA_1a+1b, and that a substantial portion of the HbA_1c in an applied sample does not elute in this fraction.