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Polisetty L, Neeharika B R. Comparative evaluation of HPLC and nephelometry for HbA1c measurement: Diagnostic agreement and clinical utility. mljgoums 2026; 20 (2) :3-6
URL: http://mlj.goums.ac.ir/article-1-1921-en.html
URL: http://mlj.goums.ac.ir/article-1-1921-en.html
1- Department of Biochemistry, Mamata Academy of Medical Sciences, Bachupally, Hyderabad, India
2- Department of Biochemistry, Mamata Academy of Medical Sciences, Bachupally, Hyderabad, India ,brneeharika@gmail.com
2- Department of Biochemistry, Mamata Academy of Medical Sciences, Bachupally, Hyderabad, India ,
Keywords: Glycated Hemoglobin, Diabetes Mellitus, Nephelometry, Ion-Exchange HPLC, Diagnostic Accuracy
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Discussion
This study aimed to compare the analytical performance and clinical applicability of nephelometry and ion-exchange HPLC for HbA1c measurement in patients with diabetes, focusing on precision, consistency, and diagnostic relevance. HbA1c provides valuable information on long-term glycaemic control and the likelihood of diabetes-related complications, making it a key marker for both diagnosing diabetes and predicting clinical outcomes (12). Our findings align with previous studies demonstrating that ion-exchange HPLC remains the benchmark method for HbA1c estimation because of its high analytical specificity and accuracy (8,11). The high correlation (r = 0.96) between nephelometry and HPLC suggests that nephelometry may be a viable alternative in settings prioritizing rapid turnaround times, provided haemoglobin variants are not a concern.
The Bland-Altman analysis demonstrated minimal systematic bias, consistent with earlier findings by Rohlfing et al. (13), and underscores the reliability of nephelometry in routine clinical use, especially when hemoglobinopathies are ruled out. Unlike many earlier studies performed under controlled laboratory conditions, the present investigation reflects operational realities in a high-prevalence, resource-constrained setting, enhancing the external validity of our findings.
ROC analysis confirmed the diagnostic advantage of HPLC (AUC = 0.94), particularly for detecting borderline HbA1c values in the prediabetic range (5.7%-6.4%), aligning with previous reports (14,15) that emphasize HPLC’s superior discriminative capacity. The slightly reduced specificity observed for nephelometry is consistent with point-of-care HbA1c studies (16), where accelerated workflows may occasionally compromise diagnostic precision. These discrepancies may arise from nephelometry's indirect measurement principle, which relies on immune complex formation and may be influenced by sample turbidity, lipid interference, or inflammatory markers. Despite minor diagnostic limitations, nephelometry demonstrated acceptable accuracy and offered operational advantages, including shorter assay time and ease of use-key considerations in high-volume laboratories and primary care settings.
Hence, although HPLC remains preferable in settings requiring rigorous analytical accuracy (e.g., diagnostic confirmation or research laboratories), nephelometry presents a viable option for initial screening and routine monitoring in high-throughput outpatient clinics. This study contributes to the current literature by presenting comparative performance data from a real-world clinical setting in India, a country with one of the highest global burdens of diabetes.”. As most prior studies have been conducted in Western populations, our findings help establish the broader applicability of both methods and emphasize the importance of balancing diagnostic precision with operational feasibility in varied healthcare environments.
Limitations
The modest sample size may limit generalizability, and potential confounders such as hemoglobinopathies or recent blood transfusions were not assessed. These factors should be addressed in future studies with larger and more diverse cohorts.
Conclusion
In conclusion, our findings demonstrate excellent agreement between ion-exchange HPLC and nephelometry for HbA1c estimation, with an ICC of 0.96 and a negligible mean difference (0.09%). While HPLC remains the reference standard because of its superior specificity, nephelometry offers a viable and faster alternative suitable for high-throughput clinical workflows, particularly in resource-constrained environments.
Acknowledgement
The authors extend their heartfelt appreciation to all participants and laboratory staff for their cooperation and assistance with data collection.
Funding Sources
Self-funded.
Ethical Statement
The study received ethical clearance from the Institutional Ethics Committee (Ref IEC/MAMS/2024/137).
Conflicts of Interest
None.
Author Contributions
All authors made intellectual contributions to the conception, design, and execution of this work.
Data Availability Statement
Not applicable.
Use of Artificial Intelligence
The authors declare that no artificial intelligence (AI) tools were used in the preparation of this manuscript.
Full-Text: (628 Views)
Introduction
With rising rates of prevalence across all age groups and regions, diabetes mellitus remains as a major public health challenge worldwide. Approximately 589 million adults aged 20-79 were living with diabetes globally in 2024 as reported by the International Diabetes Federation (IDF) Diabetes Atlas, and this number is expected to increase to 853 million by 2050 (1). A substantial majority-about 81% reside in developing countries, where diagnostic infrastructure can be variable. India, with over 101 million adults living with diabetes, ranks second only to China in disease burden (1). Early diagnosis and consistent monitoring are critical to preventing long-term complications, especially in high-prevalence settings where many individuals remain undiagnosed.
As a biomarker, glycated haemoglobin (HbA1c) plays a crucial role in identifying diabetes and monitoring chronic glycaemic control. In contrast to fasting plasma glucose and oral glucose tolerance tests, HbA1c reflects the mean blood glucose concentration over the preceding 8-12 weeks and can be measured without the need for fasting or strict timing (2-4). Both the International Expert Committee and the American Diabetes Association (ADA) recommend HbA1c for diagnostic purposes, with values of ≥ 6.5% confirming diabetes and levels between 5.7% and 6.4% identifying individuals at risk (5,3). Owing to its stability and convenience, HbA1c testing is particularly useful in outpatient and resource-limited settings.
Despite its clinical value, the reliability of HbA1c depends on the precision of the analytical method employed. The ADA specifies that only assays certified by the National Glycohemoglobin Standardization Program (NGSP) and aligned with the Diabetes Control and Complications Trial (DCCT) reference standards should be used for HbA1c measurement (3). Several analytical methods are available for this purpose, including enzymatic assays, nephelometry, immunoassays, boronate affinity methods, and ion-exchange high-performance liquid chromatography (HPLC), each differing in operational requirements, cost, and analytical accuracy (6,7).
Ion-exchange HPLC is widely regarded as the gold standard because of its excellent precision and ability to identify various hemoglobin variants. It separates hemoglobin fractions based on charge and provides detailed chromatographic profiles (8). Nephelometry, an immunoturbidimetric method, measures light scatter resulting from antigen-antibody complex formation. Although suitable for rapid, high-throughput testing, this technique may exhibit reduced analytical specificity in the presence of hemoglobin variants. According to the NGSP, immunoassay-based HbA1c measurements may be influenced by hemoglobinopathies, with the extent of interference varying between assay types (9,10).
Studies such as those by Weykamp et al. and Little et al. have shown that while both methods generally correlate well, significant method-specific biases and interferences may occur, especially in real-world settings (8,11). Method comparison studies are critical for evaluating these differences, but most are conducted in controlled environments, often without accounting for diverse population characteristics or laboratory conditions. Real-world data from high-prevalence regions such as India remain limited.
Given the clinical importance of precise HbA1c estimation for therapeutic decisions, even small variations between methods may lead to under- or over-treatment. This is particularly important in regions like India, where variability in access to certified testing platforms may result in inconsistent care.
The present study aimed to assess how well nephelometry and ion-exchange HPLC align diagnostically and to examine their practical usefulness in an Indian clinical setting. By analysing their accuracy, precision, and overall agreement, this work sought to provide meaningful comparisons between the two techniques and guide informed decisions regarding HbA1c testing methods in healthcare systems with a high disease burden and variable resource availability.
Methods
A cross-sectional observational study was conducted from October to December 2024 in the Department of Biochemistry associated with the central laboratory at a tertiary care teaching hospital in Hyderabad, Telangana, India.
A total of 50 adult patients with biochemically confirmed diabetes mellitus, including 28 males (56%) and 22 females (44%), attending the General Medicine outpatient department for follow-up, were recruited. Written informed consent was taken and participant confidentiality was maintained throughout the study. The mean age of the participants was 53.4 ± 8.2 years with a range of 35 to 70 years. Participants with pregnancy, anaemia, chronic kidney disease, liver disease, cardiovascular disease, or hypertension or who declined to give consent were excluded from the study.
Ethics approval was obtained from the Institutional Ethics Committee. Venous blood samples of approximately 5 mL were drawn from all participants using aseptic technique. HbA1c levels were estimated using two methods. The first was nephelometry, performed on the Mispa i2 analyser (Agappe diagnostics) according to the manufacturer’s instructions. The second, ion-exchange HPLC, was carried out using the Meril GluQuant HPLC system (Meril Diagnostics). Internal quality control (QC) was performed prior to sample analysis. Two levels of control (Normal and pathological) were run for each method. Mispa HbA1c control reagents (Agappe diagnostics) were used for the nephelometry assay, and Meril HbA1c control materials (Meril diagnostics) were used for the HPLC method. All control results were within manufacturer-specified acceptable ranges. HPLC recognised as the gold standard for HbA1c measurement served as the reference method in this study. For comparison, HPLC was assumed to have 100% sensitivity and specificity, and the diagnostic performance of nephelometry was evaluated against it.
Data were analysed using Microsoft Excel and SPSS version 20. Continuous variables are presented as mean ± standard deviation (SD). Pearson’s correlation coefficient (r) and the intraclass correlation coefficient (ICC) were used to assess the relationship and consistency between the two methods. Agreement between methods was evaluated using Bland-Altman analysis, reporting the mean difference and 95% limits of agreement. The diagnostic performance of the nephelometry method was determined using ion-exchange HPLC as the reference standard. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated using receiver operating characteristic (ROC) curve analysis. A diagnostic cutoff of 6.5% HbA1c was applied, following American Diabetes Association (ADA) recommendations. Statistical significance was defined as a p-value < 0.05.
Results
A total of 50 venous blood samples were analysed for HbA1c using both nephelometry and ion-exchange HPLC methods.
As shown in Table 1, the mean HbA1c values and standard deviations were closely aligned between the two methods. HPLC exhibited a slightly wider range, indicating marginally broader variability in measurement.
A high intraclass correlation coefficient (ICC) of 0.96 (p < 0.01) was observed between the two methods, indicating excellent reproducibility and inter-method agreement in HbA1c measurement (Table 2).
With rising rates of prevalence across all age groups and regions, diabetes mellitus remains as a major public health challenge worldwide. Approximately 589 million adults aged 20-79 were living with diabetes globally in 2024 as reported by the International Diabetes Federation (IDF) Diabetes Atlas, and this number is expected to increase to 853 million by 2050 (1). A substantial majority-about 81% reside in developing countries, where diagnostic infrastructure can be variable. India, with over 101 million adults living with diabetes, ranks second only to China in disease burden (1). Early diagnosis and consistent monitoring are critical to preventing long-term complications, especially in high-prevalence settings where many individuals remain undiagnosed.
As a biomarker, glycated haemoglobin (HbA1c) plays a crucial role in identifying diabetes and monitoring chronic glycaemic control. In contrast to fasting plasma glucose and oral glucose tolerance tests, HbA1c reflects the mean blood glucose concentration over the preceding 8-12 weeks and can be measured without the need for fasting or strict timing (2-4). Both the International Expert Committee and the American Diabetes Association (ADA) recommend HbA1c for diagnostic purposes, with values of ≥ 6.5% confirming diabetes and levels between 5.7% and 6.4% identifying individuals at risk (5,3). Owing to its stability and convenience, HbA1c testing is particularly useful in outpatient and resource-limited settings.
Despite its clinical value, the reliability of HbA1c depends on the precision of the analytical method employed. The ADA specifies that only assays certified by the National Glycohemoglobin Standardization Program (NGSP) and aligned with the Diabetes Control and Complications Trial (DCCT) reference standards should be used for HbA1c measurement (3). Several analytical methods are available for this purpose, including enzymatic assays, nephelometry, immunoassays, boronate affinity methods, and ion-exchange high-performance liquid chromatography (HPLC), each differing in operational requirements, cost, and analytical accuracy (6,7).
Ion-exchange HPLC is widely regarded as the gold standard because of its excellent precision and ability to identify various hemoglobin variants. It separates hemoglobin fractions based on charge and provides detailed chromatographic profiles (8). Nephelometry, an immunoturbidimetric method, measures light scatter resulting from antigen-antibody complex formation. Although suitable for rapid, high-throughput testing, this technique may exhibit reduced analytical specificity in the presence of hemoglobin variants. According to the NGSP, immunoassay-based HbA1c measurements may be influenced by hemoglobinopathies, with the extent of interference varying between assay types (9,10).
Studies such as those by Weykamp et al. and Little et al. have shown that while both methods generally correlate well, significant method-specific biases and interferences may occur, especially in real-world settings (8,11). Method comparison studies are critical for evaluating these differences, but most are conducted in controlled environments, often without accounting for diverse population characteristics or laboratory conditions. Real-world data from high-prevalence regions such as India remain limited.
Given the clinical importance of precise HbA1c estimation for therapeutic decisions, even small variations between methods may lead to under- or over-treatment. This is particularly important in regions like India, where variability in access to certified testing platforms may result in inconsistent care.
The present study aimed to assess how well nephelometry and ion-exchange HPLC align diagnostically and to examine their practical usefulness in an Indian clinical setting. By analysing their accuracy, precision, and overall agreement, this work sought to provide meaningful comparisons between the two techniques and guide informed decisions regarding HbA1c testing methods in healthcare systems with a high disease burden and variable resource availability.
Methods
A cross-sectional observational study was conducted from October to December 2024 in the Department of Biochemistry associated with the central laboratory at a tertiary care teaching hospital in Hyderabad, Telangana, India.
A total of 50 adult patients with biochemically confirmed diabetes mellitus, including 28 males (56%) and 22 females (44%), attending the General Medicine outpatient department for follow-up, were recruited. Written informed consent was taken and participant confidentiality was maintained throughout the study. The mean age of the participants was 53.4 ± 8.2 years with a range of 35 to 70 years. Participants with pregnancy, anaemia, chronic kidney disease, liver disease, cardiovascular disease, or hypertension or who declined to give consent were excluded from the study.
Ethics approval was obtained from the Institutional Ethics Committee. Venous blood samples of approximately 5 mL were drawn from all participants using aseptic technique. HbA1c levels were estimated using two methods. The first was nephelometry, performed on the Mispa i2 analyser (Agappe diagnostics) according to the manufacturer’s instructions. The second, ion-exchange HPLC, was carried out using the Meril GluQuant HPLC system (Meril Diagnostics). Internal quality control (QC) was performed prior to sample analysis. Two levels of control (Normal and pathological) were run for each method. Mispa HbA1c control reagents (Agappe diagnostics) were used for the nephelometry assay, and Meril HbA1c control materials (Meril diagnostics) were used for the HPLC method. All control results were within manufacturer-specified acceptable ranges. HPLC recognised as the gold standard for HbA1c measurement served as the reference method in this study. For comparison, HPLC was assumed to have 100% sensitivity and specificity, and the diagnostic performance of nephelometry was evaluated against it.
Data were analysed using Microsoft Excel and SPSS version 20. Continuous variables are presented as mean ± standard deviation (SD). Pearson’s correlation coefficient (r) and the intraclass correlation coefficient (ICC) were used to assess the relationship and consistency between the two methods. Agreement between methods was evaluated using Bland-Altman analysis, reporting the mean difference and 95% limits of agreement. The diagnostic performance of the nephelometry method was determined using ion-exchange HPLC as the reference standard. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated using receiver operating characteristic (ROC) curve analysis. A diagnostic cutoff of 6.5% HbA1c was applied, following American Diabetes Association (ADA) recommendations. Statistical significance was defined as a p-value < 0.05.
Results
A total of 50 venous blood samples were analysed for HbA1c using both nephelometry and ion-exchange HPLC methods.
As shown in Table 1, the mean HbA1c values and standard deviations were closely aligned between the two methods. HPLC exhibited a slightly wider range, indicating marginally broader variability in measurement.
A high intraclass correlation coefficient (ICC) of 0.96 (p < 0.01) was observed between the two methods, indicating excellent reproducibility and inter-method agreement in HbA1c measurement (Table 2).
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Table 6. Comparison of turnaround time and usability
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Discussion
This study aimed to compare the analytical performance and clinical applicability of nephelometry and ion-exchange HPLC for HbA1c measurement in patients with diabetes, focusing on precision, consistency, and diagnostic relevance. HbA1c provides valuable information on long-term glycaemic control and the likelihood of diabetes-related complications, making it a key marker for both diagnosing diabetes and predicting clinical outcomes (12). Our findings align with previous studies demonstrating that ion-exchange HPLC remains the benchmark method for HbA1c estimation because of its high analytical specificity and accuracy (8,11). The high correlation (r = 0.96) between nephelometry and HPLC suggests that nephelometry may be a viable alternative in settings prioritizing rapid turnaround times, provided haemoglobin variants are not a concern.
The Bland-Altman analysis demonstrated minimal systematic bias, consistent with earlier findings by Rohlfing et al. (13), and underscores the reliability of nephelometry in routine clinical use, especially when hemoglobinopathies are ruled out. Unlike many earlier studies performed under controlled laboratory conditions, the present investigation reflects operational realities in a high-prevalence, resource-constrained setting, enhancing the external validity of our findings.
ROC analysis confirmed the diagnostic advantage of HPLC (AUC = 0.94), particularly for detecting borderline HbA1c values in the prediabetic range (5.7%-6.4%), aligning with previous reports (14,15) that emphasize HPLC’s superior discriminative capacity. The slightly reduced specificity observed for nephelometry is consistent with point-of-care HbA1c studies (16), where accelerated workflows may occasionally compromise diagnostic precision. These discrepancies may arise from nephelometry's indirect measurement principle, which relies on immune complex formation and may be influenced by sample turbidity, lipid interference, or inflammatory markers. Despite minor diagnostic limitations, nephelometry demonstrated acceptable accuracy and offered operational advantages, including shorter assay time and ease of use-key considerations in high-volume laboratories and primary care settings.
Hence, although HPLC remains preferable in settings requiring rigorous analytical accuracy (e.g., diagnostic confirmation or research laboratories), nephelometry presents a viable option for initial screening and routine monitoring in high-throughput outpatient clinics. This study contributes to the current literature by presenting comparative performance data from a real-world clinical setting in India, a country with one of the highest global burdens of diabetes.”. As most prior studies have been conducted in Western populations, our findings help establish the broader applicability of both methods and emphasize the importance of balancing diagnostic precision with operational feasibility in varied healthcare environments.
Limitations
The modest sample size may limit generalizability, and potential confounders such as hemoglobinopathies or recent blood transfusions were not assessed. These factors should be addressed in future studies with larger and more diverse cohorts.
Conclusion
In conclusion, our findings demonstrate excellent agreement between ion-exchange HPLC and nephelometry for HbA1c estimation, with an ICC of 0.96 and a negligible mean difference (0.09%). While HPLC remains the reference standard because of its superior specificity, nephelometry offers a viable and faster alternative suitable for high-throughput clinical workflows, particularly in resource-constrained environments.
Acknowledgement
The authors extend their heartfelt appreciation to all participants and laboratory staff for their cooperation and assistance with data collection.
Funding Sources
Self-funded.
Ethical Statement
The study received ethical clearance from the Institutional Ethics Committee (Ref IEC/MAMS/2024/137).
Conflicts of Interest
None.
Author Contributions
All authors made intellectual contributions to the conception, design, and execution of this work.
Data Availability Statement
Not applicable.
Use of Artificial Intelligence
The authors declare that no artificial intelligence (AI) tools were used in the preparation of this manuscript.
Research Article: Original Paper |
Subject:
Biochemistry
Received: 2025/02/28 | Accepted: 2025/11/22 | Published: 2026/04/20 | ePublished: 2026/04/20
Received: 2025/02/28 | Accepted: 2025/11/22 | Published: 2026/04/20 | ePublished: 2026/04/20
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