HPLC vs capillary electrophoresis for HbA1c measurement is a clinically important distinction for NHS laboratory professionals and clinicians alike. HbA1c — glycated haemoglobin — is the cornerstone of diabetes diagnosis and monitoring in the UK, reported in mmol/mol using IFCC standardisation. Both high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are widely used, IFCC-traceable methods that deliver comparable accuracy in routine practice. However, their differing physical principles mean they handle haemoglobin variants and interferences differently — a critical consideration in a diverse UK population where haemoglobinopathies are prevalent.

How HbA1c Is Measured in NHS Laboratories

NHS laboratories measure HbA1c using IFCC-standardised methods — primarily HPLC or capillary electrophoresis — reported in mmol/mol, with analysers required to be UKCA- or CE-marked, IFCC-traceable, and enrolled in UK NEQAS.

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HbA1c — glycated haemoglobin — reflects average blood glucose levels over the preceding two to three months and is a cornerstone of diabetes diagnosis and monitoring. In NHS laboratories, HbA1c measurement must meet stringent quality standards to ensure clinical decisions are based on reliable data. The test quantifies the proportion of haemoglobin that has been irreversibly glycated at the N-terminal valine of the beta chain. In the UK, results are reported primarily in millimoles per mole (mmol/mol) using IFCC (International Federation of Clinical Chemistry) standardisation; the DCCT/NGSP-derived percentage may be provided secondarily for reference.

Several analytical platforms are used for HbA1c testing in the UK, with high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) being the two most widely used laboratory methods. Both techniques separate haemoglobin variants to isolate and quantify HbA1c, but they do so through fundamentally different physical principles. To be used in NHS diagnostic practice, analysers must be IVD (UKCA- or CE-) marked under the UK Medical Devices Regulations 2002 (as amended), locally verified and validated to ISO 15189 standards, and enrolled in the UK National External Quality Assessment Service (UK NEQAS) for HbA1c.

Understanding the differences between HPLC and capillary electrophoresis is important not only for laboratory professionals but also for clinicians interpreting results — particularly when haemoglobin variants or other interferences may affect accuracy. UK professional guidance from the Royal College of Pathologists (RCPath) and the Association for Clinical Biochemistry and Laboratory Medicine (ACB) provides further detail on analytical performance standards and reporting requirements.

HPLC for HbA1c: Principles, Accuracy and Clinical Use

HPLC separates haemoglobin fractions by ionic interaction on a chromatography column, offering high throughput, within-run CVs typically of 1–2%, and automated flagging of haemoglobin variants such as HbS and HbC.

High-performance liquid chromatography is currently the most widely used method for HbA1c measurement in NHS laboratories. HPLC works by passing a haemolysed blood sample through a chromatography column packed with ion-exchange resin. Haemoglobin fractions are separated based on their ionic interactions with the stationary phase, eluting at different retention times. The HbA1c fraction is identified and quantified by measuring absorbance at a specific wavelength as it passes through a detector.

Modern HPLC analysers — such as those from Bio-Rad and Tosoh — are highly automated, offering:

• High throughput, processing dozens of samples per hour

• Good reproducibility: within-run and total coefficients of variation (CV) are typically in the range of 1–2% at clinically relevant HbA1c concentrations, though performance varies by platform and concentration level — users should consult manufacturer instructions for use (IFU) and local validation data

• Simultaneous chromatographic flagging of haemoglobin variants (e.g., HbS, HbC, HbE), which can prompt further investigation

HPLC is a widely used routine method traceable to the IFCC reference measurement system (which itself uses peptide mapping and mass spectrometry). Its precision makes it particularly suitable for diagnosing diabetes at borderline HbA1c thresholds (e.g., 48 mmol/mol) and for monitoring patients with tight glycaemic targets.

However, HPLC is not without limitations. Certain haemoglobin variants can co-elute with HbA1c, potentially causing falsely elevated or falsely lowered results. Labile HbA1c and carbamylated haemoglobin (seen in renal failure) may also interfere, though the extent varies by platform. Most modern HPLC systems include flagging algorithms to alert laboratory staff when a variant is detected. It is important to note that these flags indicate the possible presence of a variant and are not a definitive haemoglobinopathy diagnosis; confirmatory testing through an appropriate haemoglobinopathy pathway is required where clinically indicated.

Capillary Electrophoresis for HbA1c: How It Works

Capillary electrophoresis separates haemoglobin fractions by charge-to-size ratio under an electric field, providing high-resolution electropherograms and clear identification of variants that may co-elute on HPLC platforms.

Capillary electrophoresis separates haemoglobin fractions using an entirely different physical principle. In CE, a haemolysed sample is introduced into a narrow capillary tube filled with an electrolyte buffer. When an electric field is applied, haemoglobin molecules migrate through the capillary at speeds determined by their charge-to-size ratio. HbA1c, which carries a different charge profile from unglycated HbA0 due to the addition of a glucose molecule, migrates at a distinct and reproducible rate, allowing accurate quantification.

The Sebia Capillarys system is the most commonly used CE platform for HbA1c in UK laboratories. Key features include:

• High resolution separation of haemoglobin fractions

• Automated sample handling with minimal manual intervention

• Clear electropherogram output, providing a visual profile of all haemoglobin fractions present

One of the notable advantages of CE is its ability to clearly resolve certain haemoglobin variants that may co-elute or interfere on HPLC platforms. Because separation is based on charge rather than ionic affinity, CE can distinguish fractions that HPLC may conflate, making it particularly useful in populations with a higher prevalence of haemoglobin variants.

Capillary electrophoresis also provides a simultaneous haemoglobin variant screen. As with HPLC, the electropherogram pattern is a screening and flagging output and is not a definitive haemoglobinopathy diagnosis; abnormal patterns should be followed up through local haemoglobinopathy diagnostic pathways. CE platforms used in NHS laboratories are traceable to the IFCC reference measurement system and enrolled in UK NEQAS proficiency schemes, confirming their suitability for routine NHS diagnostic use.

Comparing HPLC and Capillary Electrophoresis: Accuracy and Interferences

HPLC and CE deliver comparable HbA1c accuracy in patients with normal haemoglobin, but interference patterns are platform-specific; CE generally resolves more haemoglobin variants, whilst iron deficiency, haemolysis, and carbamylated haemoglobin can affect both methods.

Both HPLC and capillary electrophoresis deliver comparable accuracy for HbA1c measurement in patients with normal haemoglobin (HbAA). Method comparison studies and UK NEQAS data consistently demonstrate strong correlation between the two methods in routine clinical populations, with mean differences generally within clinically acceptable limits. For the majority of patients, either method will yield a reliable result.

The key differences emerge when haemoglobin variants are present. Common variants in the UK population include:

• HbS (sickle cell trait or disease)

• HbC (particularly in individuals of West African family origin)

• HbE (common in those of South and South-East Asian family origin)

• HbD and other rarer variants

Interference patterns are platform- and method-specific: on certain HPLC platforms, some variants co-elute with HbA1c, leading to falsely elevated or lowered results (e.g., HbS/A heterozygotes on some systems). CE generally offers better resolution of several such variants, reducing the risk of misclassification. Conversely, on some CE platforms HbE may migrate close to HbA1c on the electropherogram, potentially affecting quantification. Clinicians and laboratory staff should consult method-specific interference tables (such as those published by the NGSP) and local laboratory guidance for platform-specific detail.

Other sources of interference affecting both methods include:

• Haemolytic anaemia — shortened red cell lifespan reduces HbA1c regardless of method

• Iron deficiency anaemia — may falsely elevate HbA1c

• Carbamylated haemoglobin in chronic kidney disease — can interfere with HPLC, though the extent varies by platform and has been reduced on many modern systems

• Fetal haemoglobin (HbF) — elevated levels (e.g., in hereditary persistence of HbF) may affect results on both platforms

When interference is suspected, laboratories should use an alternative method or refer to a specialist haemoglobinopathy centre. Where HbA1c is unreliable, diagnosis of diabetes should be based on fasting plasma glucose (FPG) and/or an oral glucose tolerance test (OGTT), in line with WHO 2011 guidance and the UK position statement on HbA1c for diagnosis. Clinicians should always interpret HbA1c results in the context of the patient's clinical picture and haematological status.

NICE and Regulatory Guidance on HbA1c Testing Methods

NICE NG28 specifies HbA1c ≥48 mmol/mol as the diagnostic threshold for type 2 diabetes; NICE does not prefer HPLC over CE, provided both are IFCC-traceable, UKCA- or CE-marked, and enrolled in UK NEQAS.

In the UK, HbA1c testing for the diagnosis and monitoring of diabetes is governed by guidance from NICE, the MHRA, and the NHS England Diabetes Programme. NICE guideline NG28 (Type 2 diabetes in adults) specifies HbA1c as the primary diagnostic tool, with diagnosis confirmed at 48 mmol/mol (6.5%) or above on two separate occasions in asymptomatic individuals, or once if symptoms of hyperglycaemia are present. NICE guideline NG17 (Type 1 diabetes in adults) addresses monitoring in established type 1 diabetes; HbA1c should not be used to diagnose type 1 diabetes, nor should it be used diagnostically in children and young people, where plasma glucose criteria apply.

For an HbA1c assay to be used diagnostically in the NHS, it must be:

• Traceable to the IFCC reference measurement system

• Enrolled in UK NEQAS for ongoing external quality assurance

• UKCA- or CE-marked as an in vitro diagnostic (IVD) medical device under the UK Medical Devices Regulations 2002 (as amended), with appropriate conformity assessment

• Locally verified and validated in accordance with ISO 15189 accreditation requirements

The MHRA regulates HbA1c analysers as IVD medical devices; manufacturers must demonstrate conformity with applicable requirements before devices can be placed on the UK market. HPLC and CE platforms from major manufacturers (Bio-Rad, Tosoh, Sebia) hold appropriate UKCA or CE marking. NICE does not specify a preference for HPLC over CE or vice versa, recognising that both methods can deliver equivalent diagnostic accuracy when used appropriately.

NICE and WHO 2011 guidance are clear that HbA1c should not be used diagnostically in certain clinical situations — including pregnancy, haemolytic anaemia, haemoglobin variants, and recent blood transfusion. In these circumstances, fasting plasma glucose and/or OGTT are the appropriate diagnostic tests. Fructosamine may be considered for monitoring (not diagnosis) in selected patients where HbA1c is persistently unreliable. Continuous glucose monitoring (CGM) is not recommended as a diagnostic test for diabetes.

Choosing the Right Method for Reliable HbA1c Results

Method choice is typically determined by the local laboratory platform; clinicians should consider haemoglobinopathy history, anaemia, and renal impairment, and contact the laboratory directly if interference is suspected or results are discordant.

For most patients attending NHS primary or secondary care, the choice between HPLC and capillary electrophoresis will be determined by the local laboratory's available platform rather than a clinical decision made at the point of care. Both methods are fit for purpose in routine practice, and clinicians can generally trust results from either when the patient has normal haemoglobin and no confounding conditions.

However, clinical context matters. When ordering or interpreting an HbA1c, clinicians should consider:

• Family origin and haemoglobinopathy history — individuals of African, Caribbean, South Asian, or Mediterranean family origin, or those with a known or suspected haemoglobinopathy, have a higher prevalence of haemoglobin variants that may affect results; prior haemoglobin screening results should be reviewed where available

• Haematological conditions — anaemia, haemolysis, or recent transfusion can render HbA1c unreliable regardless of method

• Renal impairment — carbamylated haemoglobin in advanced CKD may interfere, particularly with some HPLC platforms

• Unexpectedly discordant results — if HbA1c does not align with self-monitored glucose readings or clinical presentation, interference should be suspected

When interference is identified or suspected, the laboratory should be contacted directly. Most NHS laboratories will either repeat the sample on an alternative platform (e.g., switching from HPLC to CE or vice versa) or refer to a specialist haemoglobinopathy laboratory for confirmatory testing. Laboratory scientists can advise on method-specific interference and the most appropriate course of action.

From a patient safety perspective, clinicians should not delay diabetes management while awaiting method clarification if clinical evidence of hyperglycaemia is clear. Where HbA1c is unreliable or unsuitable, fasting plasma glucose and/or OGTT should be used for diagnosis, in line with NICE NG28 and WHO 2011 guidance. Fructosamine may be considered for monitoring in selected cases where HbA1c remains persistently unreliable, but is not a diagnostic test for diabetes. Open communication between clinicians and laboratory scientists remains essential to ensuring that HbA1c results are interpreted accurately and that patients receive appropriate, timely care.

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