Stability of glycated haemoglobin (HbA1c) measurements from whole blood samples kept at -196°C for seven to eight years in The Malaysian Cohort study

Abstract

Objective: Glycated haemoglobin (HbA1c) is a standard indication for screening type 2 diabetes that also has been widely used in large-scale epidemiological studies. However, its long-term quality (in terms of reproducibility) stored in liquid nitrogen is still unknown. This study is aimed to evaluate the stability and reproducibility of HbA1c measurements from frozen whole blood samples kept at -196°C for more than 7 years.

Methods: A total of 401 whole blood samples with a fresh HbA1c measurement were randomly selected from The Malaysian Cohort's (TMC) biobank. The HbA1c measurements of fresh and frozen (stored for 7-8 years) samples were assayed using different high-performance liquid chromatography (HPLC) systems. The HbA1c values of the fresh samples were then calculated and corrected according to the later system. The reproducibility of HbA1c measurements between calculated-fresh and frozen samples was assessed using a Passing-Bablok linear regression model. The Bland-Altman plot was then used to evaluate the concordance of HbA1c values.

Results: The different HPLC systems highly correlated (r = 0.99) and agreed (ICC = 0.96) with each other. Furthermore, the HbA1c measurements for frozen samples strongly correlate with the corrected HbA1c values of the fresh samples (r = 0.875) with a mean difference of -0.02 (SD: -0.38 to 0.38). Although the mean difference is small, discrepancies were observed within the diabetic and non-diabetic samples.

Conclusion: These data demonstrate that the HbA1c measurements between fresh and frozen samples are highly correlated and reproducible.

Keywords: HbA1c; frozen whole blood; preanalytical; reproducibility; stability.

FIGURE 1

Scatterplot of calculated‐fresh versus frozen samples. The dashed line is the 45‐degree “line of agreement”, while the solid line is the Passing‐Bablok regression line with an equation of y = 0.82x + 1.11. The black circles and grey triangles represent subjects with and without diabetes, respectively.

FIGURE 2

Bland–Altman plot of differences in HbA1c measurements in calculated‐fresh and frozen samples. *denotes the fresh HbA1c value calculated using the linear regression formula. Horizontal lines were drawn for zero difference as reference (solid line), mean HbA1c difference (dashed line), and standard deviation (dotted lines). The black circles and grey triangles represent subjects with and without diabetes, respectively.