Effect of HbE trait on measurement of HbA1c by three different methods.

Sir, HbA1c testing is mainly used for monitoring glycaemic control in patients with diabetes. However, the World Health Organization now recommends that HbA1c can be used as a diagnostic test for diabetes, provided that stringent quality assurance procedures are in place and assays are standardised to criteria aligned to the international reference values. Laboratories use many different methods for measuring A1C, but some of these methods can give inaccurate results when the patient has a variant haemoglobin. The effect of common haemoglobin variants (HbS, HbE, HbC and HbD) on HbA1c measurement has been previously studied. Descriptions of the method-specific analytical interferences of these Hb variants on A1C measurement are available on the National Glycohemoglobin Standardization Program (NGSP) website. In an earlier study performed in our laboratory, a positive bias of 10% was reported when glycated haemoglobin of patients with haemoglobin (Hb) E trait was measured using Bio-Rad Variant II Turbo 2.0 [cation-exchange high performance liquid chromatography (HPLC) method; Bio-Rad, USA] compared to boronate affinity HPLC method. This was attributed to insufficient separation of A0 peak from HbE peak. But recently, Bio-Rad Variant II Turbo 2.0 introduced a new resin lot 11840 and dropline that separates HbA2 from HbA0 in the chromatogram. So it is expected that there should be no interference by the presence of common Hb variants like D and E which fall in the A2 region. Recently enzymatic HbA1c assay has been introduced in the Bio Majesty (BM) 6010/C automated chemistry analyser (JEOL, Japan). In this method, blood cells were first haemolysed, and haemoglobin was digested with protease to yield fructosyl amino acid. Fructosyl amino acid oxidase acts on the fructosyl amino acid and generates hydrogen peroxide, which reacts with chromogens in the presence of peroxidase. Capillary electrophoretic method uses the charge difference between HbA1c and other Hb fractions. Separation is achieved via a high-voltage electrical field and electro-osmotic flow. The Capillarys 2 Flex Piercing instrument (Sebia, France) uses capillary electrophoresis method and it separates normal and abnormal (or variants) levels of haemoglobin in the following order, from cathode to anode: A2/C, E, S, D, F, A0, other haemoglobins (including minor HbA1) and then A1C. However, few reports are available about the effect of HbE on HbA1c measurement by these three methods. In our routine analysis of HbA1c by the ion exchange method, the most common variant observed is HbE. Hence, we evaluated the effect of HbE trait on the measurement of HbA1c by Bio-Rad Variant II Turbo 2.0 with the new resin lot 11840, Capillarys 2 Flex Piercing for HbA1c, and enzymatic method: BM Test HbA1c on JCA BM 6010/C. Boronate affinity method (Premier Hb9210; Trinity Biotech, USA) was used as the comparison method. Samples that showed the presence of variant window when HbA1c was measured by Bio-Rad variant II turbo 2.0 were collected over a period of 3 months and were stored at 808C until further analysis. These samples were screened for