The gold standard method for HbA1c measurement underpins accurate diabetes diagnosis and long-term glycaemic monitoring across the NHS. HbA1c, or glycated haemoglobin, reflects average blood glucose over the preceding two to three months, making it a uniquely valuable clinical biomarker. Understanding which analytical methods are used, how they are standardised, and what factors can distort results is essential for clinicians and patients alike. This article explains the IFCC reference measurement procedure, the role of ion-exchange HPLC in routine NHS laboratories, relevant NICE guidance, and the physiological variables that can affect HbA1c accuracy.

What Is HbA1c and Why It Matters in Diabetes Care

HbA1c reflects average blood glucose over two to three months, making it the preferred long-term glycaemic marker for diagnosing type 2 diabetes (≥48 mmol/mol) and monitoring glycaemic control per NICE guidelines NG28 and NG17.

HbA1c, or glycated haemoglobin, is a form of haemoglobin that becomes chemically linked to glucose through a process called glycation. Because red blood cells have a lifespan of approximately 120 days, the HbA1c level reflects average blood glucose concentrations over the preceding two to three months. This makes it a uniquely valuable biomarker — unlike a fasting glucose reading, which captures only a single moment in time, HbA1c provides a broader picture of glycaemic control.

In clinical practice, HbA1c serves several important functions:

• Diagnosis of type 2 diabetes: A level of 48 mmol/mol (6.5%) or above, confirmed on a second sample if the person is asymptomatic, is diagnostic. This threshold derives from WHO 2011 guidance and has been adopted across the UK by NHS England, Public Health England (now UKHSA), and Diabetes UK.

• Monitoring of established diabetes: Regular HbA1c testing helps clinicians and patients assess whether treatment targets are being met. In line with NICE guidance (NG28 and NG17), HbA1c should be measured every three months until levels are stable, then every six months thereafter.

• Risk stratification: Levels between 42–47 mmol/mol (6.0–6.4%) indicate non-diabetic hyperglycaemia (NDH), also described as a high risk of type 2 diabetes, prompting lifestyle intervention and annual monitoring.

The NHS uses HbA1c as a cornerstone of diabetes management pathways. Its reliability, reproducibility, and clinical relevance have made it the preferred long-term glycaemic marker globally. Understanding how this test is measured — and what can influence its accuracy — is essential for both clinicians and patients to interpret results correctly and make informed treatment decisions.

The Gold Standard Method for Measuring HbA1c

The IFCC reference measurement procedure (IFCC RMP), using endoproteinase Glu-C digestion followed by HPLC-MS, is the definitive gold standard; ion-exchange HPLC, traceable to the IFCC RMP, is the standard routine method in UK laboratories.

The definitive reference method for HbA1c measurement is the IFCC reference measurement procedure (IFCC RMP), developed by the International Federation of Clinical Chemistry and Laboratory Medicine. This procedure uses endoproteinase Glu-C digestion of haemoglobin followed by high-performance liquid chromatography combined with mass spectrometry (HPLC-MS). It serves as the ultimate calibration anchor against which all routine HbA1c methods worldwide are benchmarked and standardised.

In routine NHS laboratory practice, the most widely used method is ion-exchange HPLC, which is traceable to the IFCC RMP. This technique separates haemoglobin variants based on their ionic charge interactions with a chromatography column, allowing precise quantification of HbA1c as a proportion of total haemoglobin. Ion-exchange HPLC is valued for its high precision, reproducibility, and — importantly — its ability to detect and flag haemoglobin variants that may interfere with results, producing a chromatogram that laboratory scientists can review.

Two other routine methods are also in use:

• Immunoassay-based methods: These use antibodies directed against the glycated N-terminal of the HbA1c beta chain. They are widely used on point-of-care devices and some hospital biochemistry platforms. Susceptibility to interference from haemoglobin variants varies by manufacturer and platform; some immunoassays may not flag the presence of variants.

• Boronate affinity chromatography: This method measures total glycated haemoglobin and is generally less affected by common haemoglobin variants. However, because it does not separate individual haemoglobin fractions, it cannot flag variant haemoglobins on a chromatogram, which may limit clinical interpretation in affected individuals.

The key distinction to bear in mind is that the IFCC RMP is the definitive reference, whilst ion-exchange HPLC is the predominant routine method in UK laboratories — both are IFCC-traceable, but they serve different roles within the standardisation framework.

How HPLC and IFCC Methods Are Used in NHS Laboratories

NHS laboratories use automated ion-exchange HPLC analysers calibrated to IFCC-traceable reference materials, reporting results in mmol/mol and participating in UK NEQAS EQA schemes to ensure consistency across trusts.

Within NHS laboratories, HbA1c testing is performed using platforms calibrated and traceable to the IFCC reference measurement procedure. Results are reported in IFCC units (mmol/mol), a standardised format adopted across the UK and Europe since 2009. This transition was coordinated by the Association for Clinical Biochemistry and Laboratory Medicine (ACB), NHS Diabetes, and the UK National External Quality Assessment Service (UK NEQAS), ensuring consistency and patient safety across all NHS settings.

Most NHS biochemistry laboratories use automated ion-exchange HPLC analysers — such as those manufactured by Bio-Rad or Tosoh — validated against IFCC-traceable reference materials. These instruments are subject to rigorous internal quality control (IQC) and external quality assurance (EQA) programmes. Participation in the UK NEQAS HbA1c EQA scheme is a standard requirement for accredited NHS laboratories, and laboratories are expected to meet ISO 15189 accreditation standards, ensuring that results are comparable across different hospital trusts and GP-linked laboratories.

Point-of-care (POC) HbA1c devices, such as those used in GP surgeries or diabetes clinics, typically employ immunoassay or boronate affinity chromatography methods. Per NICE guidance (DG20), POC devices are primarily recommended for monitoring rather than diagnosis. If a POC device is used for diagnostic purposes, it must demonstrate IFCC traceability and acceptable analytical performance, with robust IQC and participation in an appropriate EQA scheme, in line with local laboratory policy.

Key practical considerations in NHS laboratory practice include:

• Sample type: EDTA whole blood is the standard sample for HbA1c analysis.

• Turnaround time: Most NHS laboratories report results within 24–48 hours for routine requests.

• Haemoglobin variant flagging: Ion-exchange HPLC platforms automatically flag abnormal chromatograms, prompting further investigation when variants such as HbS or HbC are detected.

Accuracy, Standardisation, and NICE Guidelines for HbA1c Testing

IFCC standardisation ensures HbA1c results are clinically equivalent across UK laboratories; NICE NG28 and NG17 set treatment targets of 48 mmol/mol for most patients, with monitoring every three to six months.

The accuracy of HbA1c measurement depends on both the analytical method used and the standardisation framework underpinning it. The IFCC reference measurement procedure — involving endoproteinase Glu-C digestion followed by HPLC-mass spectrometry — provides the definitive calibration standard against which all routine methods are benchmarked. This global standardisation effort, coordinated through the IFCC, the American Diabetes Association (ADA), and the International Diabetes Federation (IDF), ensures that an HbA1c result of 48 mmol/mol in a London hospital is clinically equivalent to the same value reported in Edinburgh or Manchester.

Diagnostic thresholds are based on WHO 2011 guidance, as adopted in the UK by NHS England, UKHSA, and Diabetes UK:

• Diagnosis of type 2 diabetes: ≥48 mmol/mol, confirmed on a second sample if the person is asymptomatic.

• Non-diabetic hyperglycaemia (NDH) / high risk of type 2 diabetes: 42–47 mmol/mol, warranting lifestyle intervention and annual monitoring.

NICE guidelines (NG28 for type 2 diabetes and NG17 for type 1 diabetes) specify HbA1c treatment targets and monitoring frequency:

• Type 2 diabetes — lifestyle or non-hypoglycaemia-inducing drug therapy: target 48 mmol/mol (6.5%).

• Type 2 diabetes — insulin or sulfonylurea therapy: target 53 mmol/mol (7.0%), with individualisation based on the person's circumstances and risk of hypoglycaemia.

• Type 1 diabetes: target 48 mmol/mol, with individualised targets where clinically appropriate.

• Monitoring frequency: every three months until stable, then every six months (NICE NG28/NG17).

NICE also advises that HbA1c should not be used for diagnosis in certain circumstances — including in children, during pregnancy, or when haemoglobin variants or conditions affecting red cell turnover are present (see NICE NG3 for diabetes in pregnancy). In these situations, plasma glucose measurements (fasting plasma glucose or oral glucose tolerance test) remain the preferred diagnostic approach.

Laboratories are expected to meet analytical performance specifications in line with IFCC/NGSP criteria and UK NEQAS acceptability limits, which define acceptable bias and imprecision at clinically relevant concentrations.

Factors That Can Affect HbA1c Results and Clinical Interpretation

Iron deficiency anaemia, haemolytic anaemia, haemoglobin variants, and recent blood transfusion can cause falsely elevated or falsely low HbA1c results, requiring alternative tests such as fructosamine or plasma glucose in affected patients.

Whilst HbA1c is a robust and well-validated marker, several physiological and pathological factors can affect its accuracy, potentially leading to falsely elevated or falsely low results. Clinicians must be aware of these variables to avoid misdiagnosis or inappropriate treatment changes.

Conditions that may cause falsely elevated HbA1c:

• Iron deficiency anaemia (reduced red cell turnover prolongs haemoglobin exposure to glucose)

• Vitamin B12 or folate deficiency

• Splenectomy (increased red cell lifespan)

• Chronic kidney disease (carbamylation of haemoglobin may interfere with some assays)

• Ageing (associated with modestly higher HbA1c independent of glycaemia)

• Alcohol misuse and severe hypertriglyceridaemia (may affect some assay platforms)

Conditions that may cause falsely low HbA1c:

• Haemolytic anaemia (shortened red cell lifespan reduces glycation time)

• Acute blood loss or recent blood transfusion (introduces non-glycated donor haemoglobin or reduces overall red cell mass)

• Erythropoietin (EPO) therapy (stimulates production of younger, less-glycated red cells)

• Haemoglobin variants (e.g., HbS, HbC, HbE) — these may interfere with certain assay methods, though ion-exchange HPLC platforms are generally better at detecting and flagging this interference

• Pregnancy (physiological haemodilution and increased red cell turnover)

• Advanced liver disease and certain medications causing haemolysis (e.g., dapsone, some antiretrovirals)

In pregnancy, HbA1c is not recommended for diagnosing gestational diabetes or pre-existing diabetes; plasma glucose-based tests should be used instead, in line with NICE NG3.

It is also important to recognise that HbA1c reflects average glucose levels and does not capture glycaemic variability or hypoglycaemic episodes. For patients with significant hypoglycaemia unawareness or highly variable glucose profiles, continuous glucose monitoring (CGM) may provide complementary information.

From a patient safety perspective, if a clinician suspects that an HbA1c result does not reflect a patient's clinical picture — for example, if self-monitored blood glucose readings are consistently higher than the HbA1c would suggest — further investigation is warranted. In such cases, fructosamine measurement or CGM may offer alternative means of assessing glycaemic control. Patients should be encouraged to contact their GP or diabetes team if they notice discrepancies between their home glucose readings and their HbA1c results, or if they have recently been diagnosed with anaemia, a haemoglobin variant, or another condition that may affect the test.

Frequently Asked Questions