Impact of heterozygous hemoglobin E on six commercial methods for hemoglobin A1c measurement

Sharon Yong,Hong Liu,Sunil Kumar Sethi,Sin Ye Sim,Wan Ling Cheng,Tze Ping Loh,Tang Lin Teo,Qinde Liu,Felicia Fu Mun Chay,Qian Liu,Cindy Lye Teng Lum,Suru Chew,Siew Fong Neo,Lizhen Ong

doi:10.7717/peerj-achem.9

Abstract

Background This study examined the impact of heterozygous HbE on HbA1c measurements by six commonly used commercial methods. The results were compared with those from a modified isotope-dilution mass spectrometry (IDMS) reference laboratory method on a liquid chromatograph coupled with tandem mass spectrometer (LC-MS/MS). Methods Twenty-three leftover samples of patients with heterozygous HbE (HbA1c range: 5.4–11.6%), and nineteen samples with normal hemoglobin (HbA1c range: 5.0–13.7%) were included. The selected commercial methods included the Tina-quant HbA1c Gen. 3 (Roche Diagnostics, Basel, Switzerland), Cobas B 101 (Roche Diagnostics, Basel, Switzerland), D100 (Bio-Rad Laboratories, Hercules, CA, USA), Variant II Turbo HbA1c 2.0 (Bio-Rad Laboratories, Hercules, CA, USA), DCA Vantage (Siemens Healthcare, Erlangen, Germany) and HbA1c Advanced (Beckman Coulter Inc., Brea, CA, USA). Results With the exception of Cobas B 101 and the Variant II Turbo 2.0, the 95% confidence intervals of the Passing–Bablok regression lines between the results from the six commercial methods and the IDMS method overlapped. The latter suggested no statistically significant difference in results and hence no impact on HbA1c result despite the presence of heterozygous HbE. The method of Cobas B 101 gave positive bias at the range of concentrations examined (5.4–11.6%), while that of Variant II Turbo 2.0 gave positive bias at concentrations up to approximately 9.5%. The finding of significant positive bias in the methods of Cobas B 101 and Variant II Turbo 2.0 agrees with the observations of some previous studies, but is contrary to manufacturer’s claim indicating the absence of interference by heterozygous HbE. Our results also clearly showed the impact of heterozygous HbE across a fairly broad measurement range using a laboratory method (the Variant II Turbo 2.0). Laboratory practitioners and clinicians should familiarize themselves with prevailing hemoglobin variants in the population they serve and select the appropriate methods for HbA1c measurement.

Highlights

Hemoglobin E (HbE) is a variant hemoglobin caused by a single point mutation, resulting in glutamic acid to lysine substitution at position 26 of the beta chain of the hemoglobin (β 26 Glu→Lys)
This study examined the impact of heterozygous HbE on Hemologbin A1c (HbA1c) measurements using six commonly used commercial methods and a modified isotope-dilution mass spectrometry (IDMS) reference method
Statistical analysis The results for samples with HbE and normal hemoglobin obtained from the methods of the six commercial methods were plotted against those of the IDMS method using Passing–Bablok analysis with 95% confidence intervals obtained by bootstrapping

Summary

INTRODUCTION

Hemoglobin E (HbE) is a variant hemoglobin caused by a single point mutation, resulting in glutamic acid to lysine substitution at position 26 of the beta chain of the hemoglobin (β 26 Glu→Lys). The amino acid substitution shifts the overall molecular charge more basic, which can be detected by separative methods, such as electrophoresis and liquid chromatography. The measurement of HbA1c can be affected by the presence of hemoglobin variants, leading to spurious measurement that can adversely affect clinical decision making. This study examined the impact of heterozygous HbE on HbA1c measurements using six commonly used commercial methods and a modified isotope-dilution mass spectrometry (IDMS) reference method. The IDMS reference method has demonstrated comparability to the IFCC network (Liu et al, 2015)

MATERIALS AND METHODS

RESULTS

DISCUSSION

CONCLUSIONS