Evaluation of a Reference Material for Glycated Haemoglobin

Abstract

The use of lyophilized blood as a reference material for glycated haemoglobin was investigated with respect to IFCC criteria for calibrators and control materials. Ninety-two laboratories, using 11 methods, detected no changes in glycated haemoglobin content when the lyophilizate was stored for one year at 4 °C. Affinity chromatography, HPLC, electrophoresis and immunoassay detected no changes following 18 months storage at -84 and -20 °C. Samples for HPLC are stable at 4 °C for one year, and 5 years at -20 °C. For the other three methods, samples are stable for 5 years at 4 °C. At 4 °C, reconstituted samples are stable for 2 days (HPLC) and 7 days (other three). Lyophilization does not cause matrix effects and inhomogeneity, since mean glycated haemoglobin and reproducibility for lyophilized samples and whole blood were similar. The coefficient of variation for vial filling precision was 0.59%. We conclude that lyophilized blood samples can be used as calibrators and control materials. Their use as calibrators, following assignment of the HbA lc value by HPLC, may contribute, in the interim, to the standardized interpretation of long term diabetic control.

Glico-hemoglobina (HbA1c): aspectos clínicos e analíticos

“…Em paralelo, o uso prolongado de vitamina C e E também tem sido estudado como possível fonte de interferência negativa nas dosagens de GHb (84)(85)(86). O estresse oxidativo é apontado como uma provável causa do aparecimento das complicações do DM.…”

A history of HbA1c through Clinical Chemistry and Laboratory Medicine

“…The ultimate mission of this IFCC-WG was to design a reference method and to produce adapted reference materials. Taking advantage of previous publications reporting the preparation and evaluation of various control [81,82] or reference materials [83] for HbA 1c , this WG proposed in 1998 a candidate primary reference material, made of purified β -N-terminal glycated (HbA 1c ) and non-glycated (HbA o ) HbA [84] , which was used for establishing the internationally approved IFCC reference method for HbA 1c . This method relied on the separation and quantification by HPLC coupled to mass spectrometry (LC/MS) or capillary electrophoresis (LC/CE) of HbA o and HbA 1c N-terminal hexapeptides obtained by enzymatic digestion, and was published in 2002 in CCLM [85] .…”

Identification, characterization and determination of metal-binding proteins by liquid chromatography. A review

“…Most of the methods routinely applied by clinical laboratories to the determination of HbA 1c can be classified in two groups, as can be seen in the various intercomparison studies [87,89,90,91,92] 1) methods based on differences in charge between glycated and non-glycated haemoglobin (cation exchange chromatography and electrophoresis), and 2) methods based on structural characteristics of the glycated groups (affinity chromatography).…”

“…So far, some discrepancies among the results obtained with the different systems used in clinical laboratories have been reported [85,86,87,88,89,90,91,92]. For this reason and to achieve a uniform international standardization, the International Federation of Clinical Chemistry (IFCC) Working Group on HbA 1c Standardization, is developing a reference system for the international standardization of HbA 1c .…”

“…Despite all the advantages previously mentioned, some drawbacks of this technique have also been reported in some interlaboratory comparisons. Weykamp et al [91] indicate that, during one intercomparison study, it became apparent that lyophilized samples gave erroneous results with some of the instruments based on the use of affinity chromatography: Lyophilization raised the measured glycated haemoglobin by 1-1.5% over the original whole-blood samples. Such an effect was not observed by Mosca et al [92], but they also reported that the intra-laboratory variation was higher for those laboratories applying affinity chromatography than for those using other methods, however not providing any scientific justification.…”