Which method is described as a reference method for HbA1c testing?

• A. HPLC
• B. Immunoassay
• C. Capillary electrophoresis
• D. Affinity chromatography

Answer: (A)

High-performance liquid chromatography is the reference method because it directly separates and quantifies the fraction of hemoglobin that is glycated (HbA1c) from the non-glycated HbA0, using a well-characterized ion-exchange process. This separation provides a precise, reproducible measurement that can be traceably linked to international standards (IFCC/NGSP), making it the benchmark for HbA1c testing.

Immunoassays rely on antibodies against the glycated N-terminus, which can vary between kits and be affected by Hb variants, leading to inter-method differences. Capillary electrophoresis can separate Hb fractions but hasn’t been established as the standard reference method. Affinity (boronate) chromatography binds glycated molecules but does not provide the universally standardized, direct quantification of HbA1c relative to total Hb.