HPLC HbA1c Testing Offers More Than
a Number

To read an HbA1c chromatogram, start by checking that the baseline is stable and flat, which ensures accurate peak detection. Identify the major peaks—particularly the A1c and A0 peaks—by their retention times and shapes; they should be sharp, well-defined, and appear in the expected order. The A1c peak corresponds to the glycated hemoglobin and its area determines the reported HbA1c value. Watch for abnormal or extra peaks, which may indicate the presence of hemoglobin variants or other interferences. A clear, consistent chromatogram with properly integrated peaks supports a reliable HbA1c result.

Lipemia is characterized by sample turbidity resulting from the accumulation of lipoprotein particles⁵. Many patients with diabetes also have dyslipidemia, resulting in lipemic blood samples that can interfere with some HbA1c testing methods⁶. A 2018 study presented at the Association for Diagnostics & Laboratory Medicine (formerly AACC) showed that Bio-Rad’s HPLC systems, like the D-100 hemoglobin testing system, delivered accurate HbA1c results even at high triglyceride levels, while other methods showed negative bias. Unlike other systems, HPLC separates analytes by charge and polarity, making it highly resistant to lipemic interference. These findings highlight the importance of choosing Bio-Rad’s HPLC systems to ensure dependable HbA1c results.

Accurate HbA1c testing is essential for diabetes diagnosis and management, particularly in patients with elevated triglyceride levels (lipemia), a common condition in diabetic populations. The Bio-Rad D-100 hemoglobin testing system demonstrated no significant bias even at triglyceride levels up to 65 mmol/L, ensuring reliable and consistent results regardless of lipemia. This high tolerance to interference supports more confident clinical decision-making and reduces the risk of misdiagnosis or improper treatment adjustments. The graph illustrates average percent bias in HbA1c results across increasing triglyceride thresholds. Bio-Rad D-100, along with Sebia CAPILLARYS and Siemens Vista (Results A), maintains stable performance, while other platforms show increasing negative bias as triglyceride levels rise including Beckman Coulter and Roche (Results B), and Ortho VITROS and Abbott ARCHITECT (Results C) show increasing negative bias as lipemia increases.

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Screening for hemoglobinopathies and thalassemia in patients with diabetes is important for selecting the right HbA1c testing method and avoiding misleading results that can impact diagnosis and treatment. Hemoglobin variants can interfere with HbA1c measurement, either falsely elevating or lowering results⁷. Cation-exchange HPLC is one of the few methods that not only quantifies HbA1c accurately but also provides a chromatogram that can reveal the presence of many common variants. Immunoassays, on the other hand, do not detect variants and can deliver values without any visible indication of interference. With over 1,175 known hemoglobin variants, relying solely on numerical HbA1c values without variant detection can result in misdiagnosis or improper diabetes management. For this reason, understanding hemoglobinopathies and choosing a method like Bio-Rad’s HPLC—capable of identifying variant patterns—ensures clinical confidence. /p>

Switching to HPLC from other HbA1c testing methods is achievable but requires careful validation. HPLC offers more accurate and standardized reporting—especially in complex cases or diverse populations—making it a strong long-term choice^2,8. However, successful switching requires method validation, clinician communication, and possibly retraining for interpretation of chromatograms.

Read study that describes why not all HbA1c assays are created equally 

Read article to see comparison of testing solutions