Abstract
IntroductionThe 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 methodsStudy 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.ResultsMedian (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.ConclusionsStorage 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.
Highlights
The aim of the study was to evaluate the effect of a single freeze/thaw cycle on HbA1c concentrations measured by commercially available high performance liquid chromatography (HPLC) method
The aim of this study was to evaluate the effect of a 2–12 weeks storage period and single freeze/ thaw cycle on HbA1c concentrations measured by HPLC on Bio-Rad D-10® autoanalyzer in clinically healthy and diabetic individuals
HbA1c was assayed by ion-exchange high performance liquid chromatography (HPLC) method certified by National Glycohemoglobin Standardization Program (NGSP) and IFCC on Bio-Rad D-10® (Bio-Rad Laboratories, CA, USA) autoanalyzer with reportable range 18 - 179 mmol/mol (3.8–18.5%) and total precision coefficient of variation (CV) = 1.16%
Summary
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. Glycated haemoglobin (HbA1c) is a product of the non-enzymatic binding of glucose to N-terminal valine residues of haemoglobin’s β chains. It is considered a “gold standard” for monitoring diabetes, current guidelines of the American Diabetes Association (ADA) recommend its use as a diagnostic tool for diabetes [1]. Many in vitro diagnostic manufacturers developed automatic analyzers based on enzymatic, immunological and separation methods for HbA1c assay. According to the standardization programme of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), determination of HbA1c concentration should be performed using high performance liquid chromatography (HPLC) combined with capillary electrophoresis or mass-spectrometry [4]. Autoanalyzers and methods used in clinical practice are obliged to obtain IFCC certification
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