Which technique is used as a reference method for HbA1c measurement?

• A. TLC
• B. HPLC
• C. GC-MS
• D. Paper chromatography

Answer: (B)

The technique used as the reference method for HbA1c measurement is chosen because it can separate the glycated form of hemoglobin from the non-glycated form with exceptional precision and reproducibility. High-performance liquid chromatography accomplishes this by using a cation-exchange column that exploits differences in charge between HbA0 and HbA1c. As the sample passes through the column, HbA0 and HbA1c elute at different times, producing distinct peaks. The proportion of HbA1c is then determined by integrating the area of the HbA1c peak relative to the total hemoglobin peak, providing a highly accurate measurement that can be standardized across laboratories.

This method’s robustness and ability to be calibrated against international reference materials make it the standard for HbA1c measurement, ensuring consistency regardless of the lab or instrument. Other techniques like TLC, paper chromatography, and GC-MS do not offer the same level of separation precision and standardization required for a reference method, and immunoassays, while common in routine testing, are more prone to variability and interference.