Analytical Performance Evaluation of Newly Developed Glycated Reagents for Hemoglobin A1c Assay

The hemoglobin A1c (HbA1c) level is widely used to diagnose and monitor glycemic control in people with diabetes mellitus. ¹ The reliability of HbA1c measurement values depends on the analytical performance of a measurement system which is composed of analytical reagents and analyzer. As known, the ion-exchange high-performance liquid chromatography (HPLC) method is widely used to measure HbA1c levels in clinical laboratories; ² meanwhile, we found that most HbA1c analytical reagents (eluents, hemolysis and wash solution, calibrator set, and chromatography column) were dedicated by the analyzer’s manufacturer. Therefore, clinical laboratories need more choices for HbA1c analytical reagents to break the various limitations of dedicated reagents for special analyzer. Given the accuracy and precision requirements, the analytical performance of HbA1c analytical reagents from other manufacturers should be evaluated on related analyzer.

This study was designed to evaluate the various analytical performances of newly developed HbA1c reagents from Lirimax (Lirimax [Tianjin] Medical Technology Co, Ltd, China) used with the HA-8180V automatic glycohemoglobin analyzer (ARKRAY, Inc, Kyoto, Japan) according to the Clinical and Laboratory Standards Institute (CLSI) recommendation guidelines.^3-5 Overall, the results showed that the coefficient of variations (CVs) of intra-assay and inter-assay precision were less than 2.0% for both blood samples and quality controls, the relative deviations (RDs) of accuracy were less than ±1.0% for three levels of reference materials, and the carryover was 0.0196%, respectively. Furthermore, HbA1c measurement values showed a good linearity from 4.3% to 17.8% (23.0-171.0 mmol/mol) across the assay range (R² = 0.9993) in Figure 1a. All the above indicators met the acceptable criteria of the National Glycohemoglobin Standardization Program. ⁶

Figure 1.

(a) The linearity was evaluated by linear regression analysis to compare correlation between measurement values and target values of HbA1c. R² = 0.9994, range = 4.30%-17.80% (23.00-171.00 mmol/mol). (b) Correlation of HbA1c value measured with reagents from commercial reagents (ARKRAY) and newly developed HbA1c reagents (Lirimax). R² = 0.9996. (c) Comparison between commercial reagents (ARKRAY) and newly developed HbA1c reagents (Lirimax), Bland-Altman plot, and mean difference was -0.0260%.

In addition, HbA1c analytical reagents from Lirimax and ARKRAY were compared using 40 clinical blood samples (HbA1c = 4.30%-16.50% [23.0-157.0 mmol/mol]). The Pearson regression equation was Y = 0.9873x + 0.06472 (R² = 0.9996) in Figure 1b, and the Bland-Altman mean difference was −0.0260% in HbA1c (95% confidence interval [CI] = −0.1181% to 0.06612%) between two analytical reagents in Figure 1c. The results suggested that two HbA1c analytical reagents have a good consistency.

Moreover, the calibrator values were traceable to references materials (Cat nos GBW09181a and GBW09183a) and the accuracy of value transitivity was less than 1.0. The performance of calibrators could meet HbA1c value standardization. Meanwhile, the testing times of the chromatography column were evaluated by three lot columns; the service life was about 6500 times, more than 2000 times as claimed ARKRAY. The column showed an excellent service life. The reference interval was validated using blood samples from 120 healthy volunteers without diabetes, all of whose measured values fell in its recommended reference interval (4.30%-6.30% [23.0-45.0 mmol/mol]). The results indicated that the reference interval was reliable. For long-term stability, the analytical performance of HbA1c reagents was evaluated on the 3rd, 6th, 9th, 18th, and 24th month, respectively. The stability was also acceptable within 24 months.

In summary, the newly developed HbA1c reagents exhibited excellent precision, accuracy, linearity, a wide linearity range, and minimal carryover in HbA1c measurements, and calibrators possessed good traceability and value transfer, more testing times of chromatography column, and long-term stability. The newly developed HbA1c reagents could provide alternatives for a specific analyzer in clinical laboratories.