Unknown Peaks and Spuriously Low Values of Glycated Haemoglobin by High Performance Liquid Chromatography: A Cross-sectional Survey

Abstract

Introduction: Glycated haemoglobin (HbA1c) has humongous role both in diagnosis as well as in guiding the clinicians in making therapeutic decision in diabetic patients. There are various methods of laboratory estimation of glycated haemoglobin depending on charge and affinity. However, the methods are affected by a number of factors like haemoglobin variants, drugs and erythrocyte survival. Aim: To identify and evaluate abnormal peaks in chromatogram of High Performance Liquid Chromatography (HPLC) and compare it with boronate affinity chromatography and it also assessed the interference of elution of silent haemoglobin variants or drugs in exactness of HbA1c estimation by HPLC. Materials and Methods: A cross-sectional survey was conducted in the laboratory of Purwanchal Nagarik Samity, Kolkata, West Bengal, India, from November 2020 to August 2021, where 2500 samples were evaluated for HbA1c estimation. Abnormal variant window, unknown peaks or spuriously low HbA1c were identified in eight samples, where estimation was done by HPLC. These were processed further to investigate for the haemoglobin variants. The glycated haemoglobin in those samples was further estimated by Boronate affinity chromatography. Results: Out of the eight samples, three patients were Haemoglobin E trait, two were Haemoglobin E homozygous and one had sickel cell trait. However, two samples showed a normal chromatogram in HbA2/HbF mode. The glycated haemoglobin was affected by Haemoglobin E, sickel cell traits as well as other interferences that may cause inappropriate lowering of glycated haemoglobin. Conclusion: The results of this study and literature review suggests the progression of various micro/macrovascular complications in diabetic individuals can be arrested by maintaining normal HbA1c levels. Therefore, the laboratory consultant should meticulously take into consideration factors like plasma glucose concentration, drug history, biological variation or abnormal haemoglobinopathies. Thus, every laboratory catering to the huge burden of diabetic patients, where the prevalence of haemoglobinopathy is high, should individualise the method of HbA1c estimation.