The influence of sample freezing at - 80 °C for 2-12 weeks on glycated haemoglobin (HbA1c) concentration assayed by HPLC method on Bio-Rad D-10(®) auto analyzer.

INTRODUCTION: The aim of the study was to evaluate the effect of a single freeze/thaw cycle on HbA1c concentrations measured by commercially available HPLC method.

MATERIALS AND METHODS: Study included 128 whole blood samples collected from diabetic patients (N = 60) and healthy volunteers (N = 68). HbA1c concentrations were measured in fresh blood samples. Then samples were frozen at - 80 °C for up to 12 weeks. HbA1c was assayed by ion-exchange HPLC method on Bio-Rad D-10(®) analyzer. Variables were compared using Wilcoxon and ANOVA Kruskal-Wallis tests. Bias between HbA1c measured in fresh and frozen samples was calculated. The comparability of HbA1c concentrations was assessed by Bland-Altman plot.

RESULTS: Median (IQR) HbA1c concentration was 45.3 (36.6-61.2) mmol/mol for fresh and 45.3 (36.6-60.6) mmol/mol for frozen/thawed samples. No significant difference in HbA1c concentrations was found comparing fresh and frozen/thawed samples (P = 0.070) in the whole group, as well as in healthy and diabetic subjects. The median calculated bias between fresh and frozen/thawed samples was 0% in whole group and healthy subjects, and 1.19% in diabetic patients. No significant difference was found between the biases according to baseline HbA1c values (P = 0.150). The Bland-Altman plot analysis showed a positive bias of 0.4% (95% CI: - 2.8 - 3.7%), which indicates high compliance between HbA1c values and no relevant influence of sample freezing on clinical significance of HbA1c measurement.

CONCLUSIONS: Storage for up to 12 weeks at - 80 °C with a single freeze/thaw cycle does not affect HbA1c concentrations measured with HPLC method on Bio-Rad D-10(®) analyzer.