Mon, Jul 20, 2026
Volume 20, Issue 2 (Mar-Apr 2026)
mljgoums 2026, 20(2): 3-6 |
Back to browse issues page
Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
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 ,
Abstract: (3667 Views)
Background: Glycated haemoglobin (HbA1c) reflects estimated average blood glucose levels over a period of 2-3 months and is essential for both diagnosing and managing diabetes. High-performance liquid chromatography (HPLC), particularly ion-exchange HPLC, is widely regarded as the reference and gold standard method for HbA1c assessment, whereas nephelometry is commonly adopted in clinical laboratories because it provides faster results. This study compared the accuracy, reliability, and clinical applicability of these two methods.
Methods: A total of 50 patients diagnosed with diabetes mellitus and attending a tertiary care hospital were included in this cross-sectional study. For each participant, HbA1c levels were estimated using both ion-exchange HPLC and nephelometric techniques. Data analysis involved descriptive statistics, Pearson’s correlation, the intraclass correlation coefficient (ICC), Bland-Altman plots, and receiver operating characteristic (ROC) curve evaluation. A paired t-test was performed to determine statistical significance, and values with p < 0.05 were considered statistically significant.
Results: The mean HbA1c values obtained by HPLC (6.2% ± 1.5) and nephelometry (6.3% ± 1.4) were closely aligned. A strong correlation was observed between the two techniques (r = 0.96, p < 0.01), and the intraclass correlation coefficient also indicated excellent concordance (ICC = 0.96). Bland-Altman plotting revealed only a slight bias, with a mean difference of 0.09%. ROC curve evaluation showed that both methods exhibited good diagnostic capability, although HPLC achieved marginally higher sensitivity (90%) and specificity (92%) than nephelometry (88% and 91%, respectively). While nephelometry offered faster processing, its diagnostic accuracy was slightly lower.
Conclusion: Although HPLC demonstrated superior diagnostic accuracy and method agreement, nephelometry offers operational advantages in high-volume settings. Despite the high correlation between the two methods, their interchangeability should be approached with caution, particularly in borderline cases. Further studies with larger and more diverse cohorts, as well as evaluation of potential confounding factors such as hemoglobin variants, are warranted.
Methods: A total of 50 patients diagnosed with diabetes mellitus and attending a tertiary care hospital were included in this cross-sectional study. For each participant, HbA1c levels were estimated using both ion-exchange HPLC and nephelometric techniques. Data analysis involved descriptive statistics, Pearson’s correlation, the intraclass correlation coefficient (ICC), Bland-Altman plots, and receiver operating characteristic (ROC) curve evaluation. A paired t-test was performed to determine statistical significance, and values with p < 0.05 were considered statistically significant.
Results: The mean HbA1c values obtained by HPLC (6.2% ± 1.5) and nephelometry (6.3% ± 1.4) were closely aligned. A strong correlation was observed between the two techniques (r = 0.96, p < 0.01), and the intraclass correlation coefficient also indicated excellent concordance (ICC = 0.96). Bland-Altman plotting revealed only a slight bias, with a mean difference of 0.09%. ROC curve evaluation showed that both methods exhibited good diagnostic capability, although HPLC achieved marginally higher sensitivity (90%) and specificity (92%) than nephelometry (88% and 91%, respectively). While nephelometry offered faster processing, its diagnostic accuracy was slightly lower.
Conclusion: Although HPLC demonstrated superior diagnostic accuracy and method agreement, nephelometry offers operational advantages in high-volume settings. Despite the high correlation between the two methods, their interchangeability should be approached with caution, particularly in borderline cases. Further studies with larger and more diverse cohorts, as well as evaluation of potential confounding factors such as hemoglobin variants, are warranted.
Keywords: Glycated Hemoglobin, Diabetes Mellitus, Nephelometry, Ion-Exchange HPLC, Diagnostic Accuracy
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
References
1. International Diabetes Federation. IDF Diabetes Atlas, 11th edn. Brussels: International Diabetes Federation; 2024. Available from: https://idf.org/news/idf-diabetes-atlas-11th-edition/ [View at Publisher]
2. Rathod L, Khan S, Shubham S, Bisne N, Singh S, Kumar M, et al. A comparative evaluation of point-of-care and laboratory HbA1c testing in diabetes care: an Indian perspective. Cureus. 2024;16(9):e69956. [View at Publisher] [DOI] [PMID] [Google Scholar]
3. Association AD. 2. Classification and diagnosis of diabetes: Standards of care in diabetes -2024.Diabetes Care. 2024;47(Supplement_1):S309-S313. [View at Publisher] [DOI] [PMID] [Google Scholar]
4. Ramachandran A, Snehalatha C, Raghavan A, Nanditha A. Classification and diagnosis of diabetes. In: Holt RIG, Cockram CS, Flyvbjerg A, Goldstein BJ, editors. Textbook of Diabetes. 6th ed. Chichester: Wiley-Blackwell; 2024. p. 22-7. [View at Publisher] [DOI]
5. International Expert Committee. International Expert Committee report on the role of the A1C assay in the diagnosis of diabetes. Diabetes Care. 2009;32(7):1327-34. [View at Publisher] [DOI] [PMID] [Google Scholar]
6. Altawallbeh G, Makky VF, Saenger AK, Peters JM, Killeen AA. Evaluation of an ion-exchange HPLC device for HbA1c measurement. J Appl Lab Med. 2020;5(4):695-703. [View at Publisher] [DOI] [PMID] [Google Scholar]
7. Kurniawan LB. HbA1c as a diabetes mellitus biomarker and its methods evolution. Indones J Clin Pathol Med Lab. 2024;30(2):198-203. [View at Publisher] [DOI] [Google Scholar]
8. Weykamp C. HbA1c: a review of analytical and clinical aspects. Ann Lab Med. 2013;33(6):393-400. [View at Publisher] [DOI] [PMID] [Google Scholar]
9. National Glycohemoglobin Standardization Program (NGSP). Factors that Interfere with HbA1c Test Results [Internet]. Bethesda (MD):NGSP;2025. Available from: https://ngsp.org/factors.asp. [View at Publisher]
10. Rajan SS, Misquith A, Rangareddy H. Calculated glycosylated hemoglobin (HbA1c) compared with estimated HbA1c by nephelometry and its correlation to estimated average blood glucose (eAG). Galore Int J Health Sci Res. 2020;5(4):100-4. [View at Publisher]
11. Little RR, Rohlfing C, Sacks DB. The National Glycohemoglobin Standardization Program: over 20 years of improving HbA1c measurement. Clin Chem. 2019;65(7):839-48. [View at Publisher] [DOI] [PMID] [Google Scholar]
12. Sherwani SI, Khan HA, Ekhzaimy A, Masood A, Sakharkar MK. Significance of HbA1c test in diagnosis and prognosis of diabetic patients. Biomark Insights. 2016;11:95-104. [View at Publisher] [DOI] [PMID] [Google Scholar]
13. Rohlfing CL, Wiedmeyer H-M, Little RR, England JD, Tennill A, Goldstein DE. Defining the relationship between plasma glucose and HbA1c: analysis of glucose profiles and HbA1c in the Diabetes Control and Complications Trial. Diabetes Care. 2002;25(2):275-8. [View at Publisher] [DOI] [PMID] [Google Scholar]
14. Nathan DM, Kuenen J, Borg R, Zheng H, Schoenfeld D, Heine RJ Translating the A1C assay into estimated average glucose values. Diabetes Care. 2008;31(8):1473-8. [View at Publisher] [DOI] [PMID] [Google Scholar]
15. Prathima MB, Reshma S, Sushith, Shetty P, Janice D, Kalal BS, et al. Estimation of glycated haemoglobin by nephelometry, ion exchange resin, and high-performance liquid chromatography: a cross-sectional study. J Clin Diagn Res. 2020;14(9):BC01-BC05. [View at Publisher] [DOI] [Google Scholar]
16. Hirst JA, McLellan JH, Price CP, English E, Feakins BG, Stevens RJ, et al. Performance of point-of-care HbA1c test devices: implications for use in clinical practice—a systematic review and meta-analysis. Clin Chem Lab Med. 2017;55(2):167–80. [View at Publisher] [DOI] [PMID] [Google Scholar]
Send email to the article author
| Rights and permissions | |
 |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Contact us
Tel & Fax: (+98) 17-32450093
E-mail:
goums.ac.ir
yahoo.com