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HbA 1c  is the stable glucose adduct to the N-terminal group of the β-chain of HbA 0 . The measurement of HbA 1c  in human blood is most important for the long-term control of the glycaemic state in diabetic patients. Because there was no internationally agreed reference method the IFCC Working Group on HbA 1c  Standardization developed a reference method which is here described. In a first step haemoglobin is cleaved into peptides by the enzyme endoproteinase Glu-C, and in a second step the glycated and non-glycated N-terminal hexapeptides of the β-chain obtained are separated and quantified by HPLC and electrospray ionisation mass spectrometry or in a two-dimensional approach using HPLC and capillary electrophoresis with UV-detection. Both principles give identical results. HbA 1c  is measured as ratio between the glycated and non-glycated hexapeptides. Calibrators consisting of mixtures of highly purified HbA 1c  and HbA 0  are used. The analytical performance of the reference method has been evaluated by an international network of reference laboratories comprising laboratories from Europe, Japan and the USA. The intercomparison studies of the network showed excellent results with intra-laboratory CVs of 0.5 to 2% and inter-laboratory CVs of 1.4 to 2.3%. Possible interferences have been carefully investigated. Due to the higher specificity of the reference method the results are lower than those generated with most of the present commercial methods which currently are calibrated with unspecific designated comparison methods. The new reference method has been approved by the member societies of the International Federation of Clinical Chemistry and Laboratory Medicine and will be the basis for the future uniform standardization of HbA 1c  routine assays worldwide.

Approved IFCC Reference Method for the Measurement of HbA1c in Human Blood