Bio-Rad HPLC HbA1c testing is a cornerstone of accurate glycaemic assessment in UK clinical laboratories. Using ion-exchange high-performance liquid chromatography, Bio-Rad platforms such as the VARIANT II and D-10 separate haemoglobin fractions with high precision, delivering IFCC-standardised results in mmol/mol. Widely adopted across NHS biochemistry departments, these analysers support both the diagnosis of type 2 diabetes and long-term monitoring of glycaemic control. This article explains how Bio-Rad HPLC technology works, its clinical applications, potential interferences, regulatory requirements, and how results should be interpreted in line with current NICE and WHO guidance.

What Is HbA1c and Why Is It Measured?

HbA1c reflects average blood glucose over 2–3 months and is used to diagnose type 2 diabetes (≥48 mmol/mol) and monitor glycaemic control; it must not be used for diagnosis in pregnancy, children, or suspected type 1 diabetes.

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HbA1c, or glycated haemoglobin, is a well-established biomarker used to assess long-term glycaemic control in individuals with or at risk of diabetes mellitus. When glucose circulates in the bloodstream, it binds irreversibly to haemoglobin within red blood cells through a process known as non-enzymatic glycation. Because red blood cells have a lifespan of approximately 90 to 120 days, the resulting HbA1c level reflects average blood glucose concentrations over the preceding two to three months.

In clinical practice, HbA1c serves two primary purposes:

• Diagnosis of type 2 diabetes and non-diabetic hyperglycaemia — in line with WHO 2011 guidance (adopted in the UK), a single HbA1c result of 48 mmol/mol (6.5%) or above is diagnostic of type 2 diabetes in asymptomatic individuals when measured in an accredited laboratory; a result between 42 and 47 mmol/mol (6.0–6.4%) indicates non-diabetic hyperglycaemia (sometimes called prediabetes), for which lifestyle intervention is recommended (NICE PH38)

• Monitoring of glycaemic control — in people already diagnosed with diabetes, regular HbA1c testing guides treatment decisions and helps reduce the risk of long-term complications such as retinopathy, nephropathy, and cardiovascular disease

Unlike fasting plasma glucose or oral glucose tolerance tests, HbA1c measurement does not require the patient to fast, making it more convenient and reproducible in routine clinical settings. However, HbA1c must not be used for diagnosis in the following situations:

• Pregnancy or suspected gestational diabetes — an oral glucose tolerance test (OGTT) is required (NICE NG3)

• Suspected type 1 diabetes or acute-onset hyperglycaemia with symptoms — these presentations require same-day clinical assessment; HbA1c is unreliable and must not delay urgent evaluation

• Children and young people — HbA1c is not recommended as the primary diagnostic tool in this group

• Recent blood transfusion — renders HbA1c unreliable for up to three months

• Significant haemolytic anaemia or haemoglobinopathies — altered red cell turnover or abnormal haemoglobin variants can produce misleading results

• Recent acute illness, major surgery, or high-dose corticosteroid therapy — may transiently affect results

Accurate, standardised measurement in an ISO 15189-accredited laboratory is therefore essential, and the choice of analytical method plays a critical role in ensuring reliable results.

How Bio-Rad HPLC Technology Works for HbA1c Testing

Bio-Rad HPLC analysers use ion-exchange chromatography to separate haemoglobin fractions by charge, quantifying HbA1c and reporting results in mmol/mol in line with IFCC standardisation.

High-performance liquid chromatography (HPLC) is one of the most accurate and reproducible methods for measuring HbA1c in a laboratory setting. Bio-Rad Laboratories is a leading manufacturer of HPLC-based HbA1c analysers, with platforms such as the D-10, VARIANT II, and VARIANT II TURBO used extensively in clinical laboratories worldwide, including many NHS and private pathology services across the UK.

The principle of HPLC relies on ion-exchange chromatography. In brief:

• A haemolysed blood sample is injected into the analyser and carried through a chromatography column by a series of buffers with increasing ionic strength

• Different haemoglobin fractions — including HbA1c, HbA0, HbF, and various haemoglobin variants — are separated based on their charge and interaction with the column resin

• As each fraction elutes from the column, it passes through a photometric detector, generating a characteristic chromatogram with distinct peaks

• The HbA1c fraction is quantified and reported in mmol/mol in line with IFCC (International Federation of Clinical Chemistry) standardisation, which is the mandated reporting unit in the UK; the NGSP-derived percentage may be provided alongside if local policy permits

Bio-Rad's HPLC systems are designed to meet the requirements of the NGSP and IFCC reference networks, supporting traceability and comparability of results across laboratories. The automated nature of these platforms supports high-throughput testing, making them well-suited to busy NHS biochemistry departments.

Pre-analytical factors relevant to HPLC testing include sample stability, correct EDTA anticoagulation, and avoidance of prolonged storage at room temperature, which can increase labile HbA1c and affect results. Laboratories should follow manufacturer instructions for use (IFU) and IFCC/NGSP guidance on sample handling. The chromatographic output also provides additional diagnostic information, as the system can flag the presence of haemoglobin variants that may interfere with other testing methodologies.

Clinical Use of HPLC HbA1c Testing in UK Laboratories

Bio-Rad HPLC platforms are widely used across NHS biochemistry laboratories for both primary and secondary care HbA1c testing, with participation in UK NEQAS required to ensure analytical quality and inter-laboratory comparability.

In the UK, HbA1c testing for diagnostic purposes must be performed in an ISO 15189-accredited laboratory. Bio-Rad HPLC platforms represent a significant proportion of the installed base in NHS biochemistry and pathology laboratories. These analysers are used for both primary care referrals — where GPs request HbA1c as part of routine diabetes screening or monitoring — and secondary care settings, including diabetes clinics, endocrinology departments, and pre-operative assessments.

The UK National External Quality Assessment Service (UK NEQAS) oversees the quality assurance of HbA1c testing across participating laboratories. Laboratories using Bio-Rad HPLC systems are expected to participate in these external quality assurance schemes to demonstrate ongoing analytical performance and inter-laboratory comparability. This is particularly important given that HbA1c results directly influence clinical decisions, including the initiation or adjustment of glucose-lowering therapies.

From a workflow perspective, Bio-Rad HPLC analysers offer several practical advantages in the NHS laboratory environment:

• High throughput — capable of processing large sample volumes efficiently, reducing turnaround times

• Minimal sample preparation — whole blood samples collected in EDTA tubes can be processed directly

• Automatic flagging — the system identifies abnormal chromatograms suggestive of haemoglobin variants, prompting further investigation

• IFCC-aligned reporting — results are reported in mmol/mol, consistent with UK clinical guidelines

Point-of-care (POC) HbA1c devices are available for near-patient testing in GP surgeries and community clinics; however, most UK POC platforms use immunoassay or boronate affinity methods rather than HPLC. Importantly, POC HbA1c results must not be used for diagnosis — a confirmatory result from an ISO 15189-accredited laboratory is required before a diagnosis of diabetes or non-diabetic hyperglycaemia is made. POC testing may have a role in monitoring in some settings, but clinicians should be aware that results from different platforms may show minor variation, and where diagnostic uncertainty exists, repeat laboratory testing or alternative methods may be warranted.

Accuracy, Interference, and MHRA Considerations

Haemoglobin variants are the most significant source of interference with HPLC HbA1c testing; Bio-Rad systems flag abnormal chromatograms, and all IVD devices must comply with MHRA regulations and carry appropriate UKCA or CE marking.

HPLC is one of the most accurate methods for HbA1c quantification available in routine laboratory practice, and Bio-Rad platforms are specifically engineered to minimise common sources of analytical error. However, no method is entirely free from potential interference, and laboratory scientists and clinicians must be aware of factors that can affect result reliability.

Haemoglobin variants represent the most clinically significant source of interference. Conditions such as sickle cell trait (HbAS), HbC, HbD, HbE, and HbS disease can alter the chromatographic profile. Bio-Rad HPLC systems are generally capable of detecting and flagging many of these variants, and in some cases can still provide a valid HbA1c result. However, in the presence of certain variants — particularly HbSS or HbCC — HbA1c may be unreliable across all methods. In such cases, alternative markers of glycaemic control such as fructosamine, glycated albumin, or structured self-monitoring of blood glucose (SMBG) or continuous glucose monitoring (CGM) should be considered in discussion with a diabetes specialist. Clinicians should consult ACB/RCPath guidance on the management of HbA1c results in the presence of haemoglobin variants.

Other factors that may affect HbA1c accuracy include:

• Haemolytic anaemia — increased red cell turnover leads to falsely low HbA1c values

• Iron deficiency anaemia — may cause falsely elevated results due to prolonged red cell lifespan

• Recent blood transfusion — dilutes the patient's own haemoglobin, rendering HbA1c unreliable for up to three months

• Advanced chronic kidney disease (uraemia) — can affect glycation rates and haemoglobin integrity

• Certain medications — including dapsone and some antiretroviral agents, which may cause haemolysis or affect red cell lifespan

• High HbF levels — for example in neonates or certain haematological conditions — can affect HPLC chromatographic separation

From a regulatory standpoint, in vitro diagnostic (IVD) devices used in UK laboratories — including Bio-Rad HPLC analysers — must comply with the UK Medical Devices Regulations 2002 (as amended). Devices must carry appropriate UKCA marking (or CE marking under current transitional arrangements; laboratories should check current MHRA guidance for applicable timelines). Oversight is provided by the Medicines and Healthcare products Regulatory Agency (MHRA). Laboratories should ensure that their Bio-Rad systems are used within the manufacturer's validated parameters, that the laboratory holds ISO 15189 accreditation via UKAS, and that any device-related safety concerns are reported through the MHRA Yellow Card scheme for medical devices.

Interpreting HbA1c Results in Line with NICE Guidelines

HbA1c of 48 mmol/mol or above is diagnostic of type 2 diabetes in asymptomatic adults; results between 42–47 mmol/mol indicate non-diabetic hyperglycaemia requiring lifestyle intervention per NICE PH38.

Correct interpretation of HbA1c results is essential for safe and effective clinical management. The following NICE guidelines are relevant to HbA1c use and interpretation:

• NICE NG28 — Type 2 diabetes in adults: management

• NICE NG17 — Type 1 diabetes in adults: diagnosis and management

• NICE NG3 — Diabetes in pregnancy: management from preconception to the postnatal period

• NICE PH38 — Type 2 diabetes: prevention in people at high risk (non-diabetic hyperglycaemia)

All results should be interpreted in the context of the individual patient's clinical history and in line with WHO 2011 diagnostic thresholds as adopted in the UK.

Diagnostic thresholds (WHO/NICE-aligned, for use in accredited laboratories only):

• Below 42 mmol/mol (6.0%) — normal; no evidence of diabetes or non-diabetic hyperglycaemia

• 42–47 mmol/mol (6.0–6.4%) — non-diabetic hyperglycaemia; lifestyle intervention recommended (NICE PH38)

• 48 mmol/mol (6.5%) or above — diagnostic of type 2 diabetes in asymptomatic individuals; a repeat confirmatory test is required unless symptoms of diabetes are present

Important diagnostic exclusions: HbA1c must not be used to diagnose diabetes in pregnancy, in children and young people, or where type 1 diabetes or acute-onset hyperglycaemia is suspected. Suspected type 1 diabetes or diabetic ketoacidosis (DKA) requires same-day clinical assessment and urgent referral — do not rely on HbA1c in these situations.

Monitoring targets vary by patient group and treatment regimen. NICE NG28 recommends an HbA1c target of 48 mmol/mol (6.5%) for most people with type 2 diabetes managed by lifestyle or metformin alone, rising to 53 mmol/mol (7.0%) for those on medications that carry a risk of hypoglycaemia. Individualised targets may be appropriate for older adults, those with frailty, or individuals with significant comorbidities.

A single HbA1c result should not be used in isolation. Clinicians should consider the full clinical picture, including symptoms, fasting glucose, and risk factors. Patients should be informed of their result in plain language and advised on the implications for their health. Where results are borderline or discordant with clinical presentation, repeat testing or referral to a diabetes specialist may be appropriate. GPs and practice nurses play a central role in communicating results and supporting patients in making informed decisions about lifestyle and treatment.

Choosing an HbA1c Testing Method: What NHS Labs Should Know

NHS laboratories selecting an HbA1c platform must ensure IFCC/NGSP alignment, ISO 15189 accreditation, acceptable analytical imprecision (CV <2%), haemoglobin variant detection capability, and compliance with MHRA regulatory requirements.

When selecting an HbA1c testing platform, NHS laboratories must balance analytical performance, cost-effectiveness, throughput requirements, and compliance with national quality standards. For diagnostic purposes, HbA1c must be measured using a laboratory-based, IFCC/NGSP-aligned method in an ISO 15189-accredited laboratory. Bio-Rad HPLC systems are among the most widely validated platforms available and their adoption in UK laboratories reflects both their technical capabilities and their alignment with IFCC standardisation requirements. It should be noted, however, that HPLC is one of several method types — alongside boronate affinity chromatography and immunoassay — that can meet IFCC/NGSP performance criteria; the IFCC reference method itself is a distinct, specialist technique not used in routine laboratory practice.

Key considerations for NHS procurement and laboratory managers include:

• Analytical performance — the method should demonstrate acceptable imprecision (CV <2%) and bias relative to IFCC reference values, as assessed through UK NEQAS participation and in line with ACB/RCPath guidance

• Haemoglobin variant detection — platforms that can identify and flag variants reduce the risk of misdiagnosis in ethnically diverse populations, which is particularly relevant in many UK urban settings

• Throughput and automation — high-volume laboratories should assess whether the platform can meet demand without compromising turnaround times

• Reagent and maintenance costs — whole-life costs, including consumables, calibration, and service contracts, should be factored into procurement decisions

• Regulatory compliance — all IVD devices must meet MHRA requirements and carry appropriate UKCA marking or CE marking under current transitional arrangements; laboratories should verify current MHRA guidance on applicable timelines. ISO 15189 accreditation via UKAS is required for laboratories performing diagnostic HbA1c testing

Immunoassay-based methods are also widely used and may be more suitable for smaller laboratories. Point-of-care platforms — which typically use immunoassay or boronate affinity methods rather than HPLC — must not be used for diagnosis. Laboratories should consult the Association for Clinical Biochemistry and Laboratory Medicine (ACB) and Royal College of Pathologists (RCPath) guidance, alongside IFCC/NGSP method performance criteria and UK NEQAS requirements, when reviewing their HbA1c testing provision. Ultimately, the goal is to ensure that every patient receives an accurate, reproducible result from an accredited laboratory that supports safe and effective clinical care.

Reporting concerns: Any suspected device malfunction or safety issue with an IVD analyser should be reported to the MHRA via the Yellow Card scheme for medical devices.

Frequently Asked Questions