HbA1c (HPLC and immunoassay)

Principle

• HbA1c forms by slow, non-enzymatic glycation of the N-terminal valine of the haemoglobin β-chain (stable ketoamine).
• It reflects average glycaemia over the previous 2–3 months (red cell lifespan about 120 days); recent weeks contribute most.
• Cation-exchange HPLC separates haemoglobin fractions by charge; HbA1c elutes as a distinct peak and is expressed as a percentage of total Hb. Chromatograms also reveal variants (HbS, HbC, HbE, raised HbF).
• Immunoassay (usually turbidimetric inhibition) uses antibodies against the glycated N-terminal end of the β-chain; total Hb is measured separately and the ratio calculated.
• Other methods: boronate affinity chromatography and enzymatic assays.

Specimen

• EDTA whole blood; fasting not required. Stable several days at 2–8 °C.

Results

• Assays should be NGSP-certified and IFCC-traceable.
• <5.7% (<39 mmol/mol): normal.
• 5.7–6.4% (39–47 mmol/mol): prediabetes (ADA).
• ≥6.5% (≥48 mmol/mol): diabetes, confirmed by repeat testing unless clear hyperglycaemic symptoms.
• Common treatment target for many adults: <7% (53 mmol/mol).
• IFCC (mmol/mol) = (NGSP % – 2.15) × 10.929.

Quality control and pitfalls

• Falsely low: shortened red cell survival (haemolytic anaemia, acute blood loss), recent transfusion, pregnancy, erythropoietin therapy.
• Falsely high: iron-deficiency anaemia and other states with older red cell populations; asplenia.
• Haemoglobin variants can interfere with some methods; check the HPLC trace and choose an unaffected method.
• High HbF gives falsely low results in some methods.
• Not reliable for diagnosis in conditions that alter red cell turnover; use plasma glucose instead.

Clinical use

• Diagnosis of diabetes and long-term monitoring of glycaemic control.