Haemoglobin variants affect the accuracy of HbA1c assays in ways that can lead to significant diagnostic and monitoring errors in diabetes care. HbA1c is the standard marker for long-term glycaemic control, but its reliability depends on normal haemoglobin structure and red cell lifespan. When variants such as HbS, HbC, HbE, or HbD are present — or when conditions such as haemolytic anaemia or recent transfusion alter red cell survival — results can be falsely low or high. This article explains the mechanisms of interference, the assay methods most affected, relevant NICE and NHS guidance, and how to manage glycaemic monitoring safely in affected patients.

How Haemoglobin Variants Interfere with HbA1c Measurement

Haemoglobin variants interfere with HbA1c assays by altering glycation sites, shortening red cell lifespan, or causing co-elution with the HbA1c fraction, producing falsely low or high results depending on the variant and analytical method.

HbA1c (glycated haemoglobin) is a cornerstone of diabetes diagnosis and long-term glycaemic monitoring. It reflects average blood glucose concentration over the preceding 8–12 weeks by measuring the proportion of haemoglobin A that has been non-enzymatically glycated at the beta-chain N-terminal valine residue, though the result is weighted towards the most recent 4–6 weeks rather than being an even average across the full period. This process is directly proportional to ambient glucose levels, making HbA1c a reliable and convenient marker under normal circumstances.

However, the accuracy of HbA1c assays depends critically on the assumption that the patient has a normal haemoglobin structure and red cell lifespan. When haemoglobin variants are present — such as HbS, HbC, HbE, or HbD — the structural differences in the globin chains can interfere with the assay in several ways:

• Altered glycation sites: Amino acid substitutions near the glycation site can reduce or enhance glycation, producing falsely low or high results.

• Reduced red cell lifespan: Many haemoglobinopathies cause haemolytic anaemia, shortening red cell survival and thereby reducing the time available for glycation, leading to spuriously low HbA1c values.

• Co-elution or misidentification: Some variants co-elute with the HbA1c peak in chromatography-based methods, causing false elevation.

• Assay-specific interference: Different analytical platforms respond differently to the same variant, meaning a result may appear normal on one method but be unreliable on another.

In addition to structural variants, two further sources of interference are clinically important:

• Elevated fetal haemoglobin (HbF): Conditions associated with persistently raised HbF — such as hereditary persistence of fetal haemoglobin (HPFH) or delta-beta thalassaemia — can interfere with certain assay methods, as HbF is glycated differently from HbA and may co-elute with or displace the HbA1c fraction depending on the platform used.

• Recent red cell transfusion: Transfused red cells from a donor without diabetes will dilute the patient's own glycated haemoglobin, producing a spuriously low HbA1c. This effect can persist for up to 2–3 months following transfusion, rendering HbA1c uninterpretable during this period.

Understanding these mechanisms is essential for clinicians interpreting HbA1c results in patients from populations where haemoglobinopathies are prevalent, as misinterpretation can lead to under- or over-treatment of diabetes. Authoritative guidance on method-specific interferences is available from the NGSP (National Glycohemoglobin Standardization Program), the IFCC Task Force on HbA1c, and the Royal College of Pathologists (RCPath) and Association for Clinical Biochemistry (ACB) UK position statements.

Common Hb Variants Encountered in UK Clinical Practice

HbS, HbC, HbE, HbD, beta-thalassaemia trait, and raised HbF are the most clinically significant variants affecting HbA1c accuracy in UK practice, with prevalence highest in individuals of African, Caribbean, and South or South-East Asian heritage.

The United Kingdom has a diverse population, and haemoglobin variants are encountered with increasing frequency in clinical practice. The NHS Sickle Cell and Thalassaemia Screening Programme identifies many of these variants at birth or during antenatal screening, but adults may present without a prior diagnosis.

The most clinically significant variants affecting HbA1c accuracy include:

• HbS (sickle cell trait or disease): Caused by a glutamic acid-to-valine substitution at position 6 of the beta-globin chain. HbS is among the most common variants in the UK, particularly in individuals of African, Caribbean, or Middle Eastern heritage. In sickle cell disease (HbSS), severe haemolysis markedly shortens red cell lifespan, producing falsely low HbA1c. In sickle cell trait (HbAS), red cell lifespan is typically normal; however, method-dependent interference can still occur and should be checked against NGSP interference tables for the specific platform in use.

• HbC: Found predominantly in West African populations. HbC trait (HbAC) may cause mild method-dependent interference, whilst HbCC disease causes more significant haemolysis and a greater risk of spuriously low results.

• HbE: Common in South and South-East Asian populations. HbE is associated with a thalassaemic phenotype and can cause mild haemolytic anaemia, with interference varying by assay method.

• HbD: Less common but found in South Asian and some Middle Eastern populations; may interfere with certain assay platforms.

• Beta-thalassaemia trait: Whilst not a structural variant, reduced HbA production alters the ratio of HbA to HbA1c. Importantly, HbA1c may remain reliable in beta-thalassaemia trait if red cell lifespan is normal and the analytical method is not affected by the altered haemoglobin pattern; interference is method-specific and should not be assumed in all cases.

• Raised HbF (HPFH and delta-beta thalassaemia): Persistently elevated HbF can interfere with certain HPLC and immunoassay platforms. Clinicians should be aware of this possibility in patients with unexpectedly discordant results.

• Alpha-thalassaemia traits: These generally have a lesser impact on HbA1c than beta-chain variants, but method-specific interference has been reported and should be considered when results are inconsistent with clinical findings.

Clinicians should be aware of the ethnic background and haematological history of their patients when requesting or interpreting HbA1c, particularly in areas of the UK with high prevalence of these conditions, such as London, Birmingham, and Leicester. The NHS Sickle Cell and Thalassaemia Screening Programme provides useful epidemiological context and clinical pathways for affected individuals.

Which HbA1c Assay Methods Are Most Affected

HPLC and capillary electrophoresis are most susceptible to variant interference through co-elution; boronate affinity chromatography is least affected by structural variants but remains unreliable when red cell lifespan is shortened.

Several analytical methods are used to measure HbA1c, and their susceptibility to interference from haemoglobin variants differs considerably. Understanding these differences helps laboratories and clinicians select the most appropriate method or interpret results with appropriate caution. The NGSP and IFCC Task Force on HbA1c maintain up-to-date, method-specific interference tables that laboratories and clinicians should consult when a variant is known or suspected.

Ion-exchange high-performance liquid chromatography (HPLC) is the most widely used method in UK laboratories. It separates haemoglobin fractions by charge. Variants such as HbS and HbC can co-elute with the HbA1c fraction or produce abnormal chromatograms, flagging the result as unreliable. Most modern HPLC analysers generate a warning flag and a chromatographic trace when a variant is detected; reviewing the trace is an important step in identifying interference.

Immunoassay methods use antibodies directed against the glycated N-terminal peptide of the beta-chain. These are generally less affected by variants that alter the beta-chain structure away from the antibody-binding site, but HbS and HbC — which involve substitutions at position 6 — can still cause interference depending on the specific antibody used.

Capillary electrophoresis separates haemoglobin fractions by charge in a capillary tube and is similarly susceptible to co-migration of variants with HbA1c. Many capillary electrophoresis platforms produce electropherograms that can aid in the visual identification of variant peaks, and these should be reviewed alongside any numerical result.

Boronate affinity chromatography measures total glycated haemoglobin regardless of the specific chain affected. It is considered the least susceptible to structural variant interference, as it does not rely on charge-based separation. However, it remains affected by conditions that alter red cell lifespan, such as haemolytic anaemia or recent transfusion. Some guidelines recommend this method when structural variants are known to be present, though it is not universally available.

UK medical laboratories are accredited by the United Kingdom Accreditation Service (UKAS) to ISO 15189 and typically participate in UK NEQAS (National External Quality Assessment Service) external quality assessment (EQA) schemes for HbA1c. Laboratories are expected to flag results where variant interference is suspected and to add interpretive comments to the report. Clinicians should always review accompanying laboratory comments rather than acting on the numerical result alone. Known interferences for specific platforms are detailed in manufacturers' instructions for use (IFUs) and in NGSP and IFCC resources.

NICE and NHS Guidance on HbA1c Testing in Haemoglobinopathies

NICE advises that HbA1c should not be used for diabetes diagnosis or monitoring when haemoglobin variants or haemolysis are present; fasting plasma glucose, OGTT, fructosamine, or CGM should be used instead.

NICE guidance on the diagnosis and management of type 2 diabetes (NG28) and type 1 diabetes (NG17), along with the NICE guideline on diabetes in pregnancy (NG3), acknowledges that HbA1c may not be reliable in certain clinical circumstances, including haemoglobinopathies and haemolytic anaemias.^[7] In these situations, NICE advises that alternative markers of glycaemic control should be used. For diagnostic purposes, fasting plasma glucose (FPG) or an oral glucose tolerance test (OGTT) should be considered when HbA1c is unreliable; for ongoing monitoring, self-monitored blood glucose (SMBG), continuous glucose monitoring (CGM), or fructosamine may be appropriate depending on clinical context.

The NHS and the Joint British Diabetes Societies (JBDS) have also issued practical guidance highlighting that:

• HbA1c should not be used for diagnosis of diabetes in individuals with known haemoglobin variants or conditions causing haemolysis, as results may be falsely low and lead to missed diagnoses.

• Monitoring HbA1c in established diabetes is similarly unreliable in these patients, and clinicians should document the limitation clearly in the patient record.

• Laboratory reports should include interpretive comments when a haemoglobin variant is detected, and clinicians are advised not to act on flagged results without further assessment.

Known interferences for specific HbA1c analysers are detailed in manufacturers' instructions for use (IFUs) and in NGSP and IFCC interference resources; laboratories are responsible for validating their methods against relevant variants and communicating limitations to requesting clinicians.

In UK practice, laboratories using HPLC or capillary electrophoresis platforms are well placed to detect variant interference through chromatographic traces or electropherograms. When interference is confirmed or strongly suspected, the laboratory should recommend an alternative test and communicate this clearly to the requesting clinician. The RCPath and ACB have published UK-specific position statements on HbA1c measurement and interpretation that provide further practical guidance for laboratories and clinicians.

Alternative Markers for Monitoring Glycaemic Control

Fructosamine, glycated albumin, and CGM are the principal alternatives to HbA1c when haemoglobin variants render it unreliable, with CGM offering real-time glucose data and metrics such as time in range.

When HbA1c is unreliable due to haemoglobin variants, haemolytic conditions, or recent transfusion, alternative biomarkers must be used to assess glycaemic control. Each has its own advantages and limitations, and the choice should be guided by clinical context and local laboratory availability.

Fructosamine measures glycated serum proteins, predominantly albumin, reflecting average glucose over the preceding 2–3 weeks. It is not affected by haemoglobin variants and is widely available in NHS laboratories. However, it is influenced by conditions that alter serum protein levels, such as nephrotic syndrome, liver disease, or thyroid dysfunction, and lacks the long-term standardisation of HbA1c.

Glycated albumin is a more specific measure of albumin glycation and reflects a 2–3 week average glucose. It is less affected by protein turnover than total fructosamine and is used in some specialist centres, though it is not yet routinely available across all NHS trusts.

Continuous glucose monitoring (CGM) has become increasingly important in this context. CGM devices measure interstitial glucose in real time and can generate metrics such as:

• Time in range (TIR): The percentage of time glucose remains within 3.9–10.0 mmol/L, as defined by international consensus (Advanced Technologies and Treatments for Diabetes, ATTD)

• Time above range (TAR) and time below range (TBR)

• Glucose management indicator (GMI): An estimated HbA1c equivalent derived from CGM data — this is an approximation and should not be used as a direct substitute for a laboratory HbA1c result, particularly when HbA1c is known to be unreliable

NICE NG17 recommends CGM for all adults with type 1 diabetes.^[6] NICE NG28 sets out specific criteria under which CGM or flash glucose monitoring may be offered to people with type 2 diabetes, including those on insulin with recurrent hypoglycaemia or an inability to self-monitor. For patients with haemoglobinopathies and diabetes, CGM offers a particularly valuable alternative, providing granular glycaemic data without reliance on red cell-based markers.

When to Refer and How to Interpret Results Accurately

Clinicians should refer to haematology when a haemoglobin variant is newly suspected, and should use fructosamine or CGM for glycaemic monitoring in all patients with known haemoglobinopathies or recent transfusion.

Accurate interpretation of HbA1c in patients with haemoglobin variants requires a collaborative approach between primary care, diabetes teams, and haematology services. Clinicians should be alert to the possibility of a haemoglobin variant in any patient whose HbA1c result appears inconsistent with their self-monitored blood glucose readings, CGM data, or clinical presentation.

Referral triggers and practical steps include:

• Unexpectedly low HbA1c in a patient with persistently elevated glucose readings — consider haemolysis or a haemoglobin variant as a cause.

• Laboratory flag or abnormal chromatogram or electropherogram — do not act on the numerical result; contact the laboratory for clarification and request an alternative test.

• Known haemoglobinopathy — document this clearly in the patient record and establish an agreed monitoring strategy using fructosamine or CGM from the outset.

• Newly diagnosed or suspected haemoglobinopathy — refer to haematology for confirmation and genotyping, and inform the diabetes team.

• Recent red cell transfusion (within the preceding 2–3 months) — HbA1c is unreliable during this period regardless of haemoglobin variant status; use fructosamine or CGM for glycaemic assessment until sufficient time has elapsed.

• Pregnancy in a patient with a haemoglobin variant — urgent referral to a combined obstetric diabetes and haematology clinic is recommended, as glycaemic targets are tighter and HbA1c unreliability poses greater risk. Relevant guidance is available from NICE NG3, the RCOG, and JBDS guidance on pregnancy glycaemic targets and CGM use.

When interpreting results, clinicians should always consider the whole clinical picture: symptoms, self-monitored glucose logs, CGM data, and any haematological indices suggesting haemolysis (e.g., raised bilirubin, low haptoglobin, elevated reticulocyte count). A multidisciplinary approach — involving the GP, diabetes specialist nurse, diabetologist, and haematologist where appropriate — ensures that patients with haemoglobin variants receive safe, accurate, and individualised diabetes care. Clear documentation and patient education about why standard HbA1c monitoring may not apply to them are equally important components of safe practice.

Frequently Asked Questions