Letter to the Editor
Influence of hemoglobin E on measurement of hemoglobin A1c by immunoassays

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Abstract

Background

Many assays used for HbA1c measurement can be interfered by hemoglobin variants. Hemoglobin E is one of the most common variant. Effect of HbE on high performance liquid chromatography was widely reported but not on immunoassay.

Objective

To determine the effect of hemoglobin E on HbA1c values by immunoassay methods.

Material and methods

Two immunoassays were used for measurement of HbA1c in samples with hemoglobin type as A2A (normal controls, n = 60), EA (heterozygous hemoglobin E, n = 151), and EE (homozygous hemoglobin E, n = 43). Results within each assay and each hemoglobin typing group were compared. Mann–Whitney U test, Pearson's correlation and linear regression analysis were used.

Results

There were significant differences of HbA1c values between normal controls and hemoglobin E-contained samples. Correlation between two assays was worse in the presence of hemoglobin E comparing to normal controls.

Conclusion

Hemoglobin E can affect the immunoassays used for HbA1c measurement. Further studies are needed to understand the mechanism of interference.

Introduction

Hemoglobin E (HbE) is the most common hemoglobin variant in Southeast Asia and the second most prevalent hemoglobin variant worldwide. Its average frequency is 13% and may reach 50–60% in some part of Thailand, Laos, Cambodia, and Vietnam [1]. To date, numbers of assays for HbA1c measurement are commercially available. Effect of HbE on assays for HbA1c measurement such as ion-exchange HPLC is widely reported but there are only few data on immunoassays [2], [3], [4], [5], [6]. Therefore the objective of this study is to evaluate effect of HbE on commercial HbA1c assays in our hospital.

Section snippets

Materials and methods

Three hundred and eighteen samples were collected in EDTA tubes. Hemoglobin typing was performed on VARIANT™ II β Thalassemia Short Program (BIO-RAD; California, USA). Samples with hemoglobin type as normal control (A2A), HbE heterozygote (AE) and HbE homozygote (EE) were then analyzed for HbA1c using two immunoassays, COBAS INTEGRA (Roche Diagnostics, USA) and Dimension RxL HbA1c kit (Dade Behring, USA). Results from samples with HbA1c level over 6% were excluded. Mann–Whitney U test and

Results

There were 60 normal controls, 151 HbE heterozygotes and 43 HbE homozygotes. Significant differences of HbA1c values between normal controls and EA or EE groups were found (p < 0.05) regardless of HbA1c assays (Fig. 1). There was no correlation between HbE level and HbA1c from both immunoassay (p < 0.001). In addition, poorer correlation of HbA1c between two immunoassays were found in the presence of HbE comparing with normal controls (p < 0.001).

Discussion and conclusion

Effect of HbE on accuracy and comparability of immunoassays for HbA1c measurement was presented here. Since abnormal of beta globin chain of HbE is not nearby the N-terminal, it should not interfere with interaction of monoclonal antibody used in an immunoassay. Furthermore there was no correlation between amount of HbE and HbA1c values. Thus the effect of HbE on immunoassay may not directly depend on the amount of HbE. Other biological factors must be considered, for example, shortening life

Conflict of interest

The authors declare that they have no conflict of interest.

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