Effect of hemoglobin F and A₂ on hemoglobin A_1c determined by cation exchange high-performance liquid chromatography

Introduction

A normal hemoglobin molecule is composed of two dissimilar pairs of polypeptide chains (alpha and non-alpha) called globins. The combination of two alpha chains and two non-alpha chains determines the hemoglobin type. Hemoglobin (Hb) A (a₂β₂) is the major Hb in adult persons, accounting for more than 96% of total Hb. The small part of total Hb constitutes HbA₂ (a₂δ₂) and fetal Hb (HbF) (α₂γ₂) that do not have β-globin chain. The problems in β-globin chain production, the production of δ-globin and γ-globins may be increased, resulting in higher levels of HbA₂ and HbF. Higher levels of HbA₂ and HbF are biological markers for the β-thalassemia [1]. Carriers of β-thalassemia according to World Health Organization estimates are approximately 1%–5% of the world’s population [2]. Also, they are a very common patient group in Turkey, because it originated in the Mediterranean and in the Middle East [3]. In addition, increases in HbF can be observed in some conditions such as hereditary persistence of fetal Hb and some drug treatments [4], [5].

Glycated hemoglobin (HbA_1c) is a marker to be correlated with complications of diabetes mellitus (DM). It has been recommended for diagnosing DM as well as for specific treatment goals in this disease [6]. Therefore, accurate measurement of HbA_1c is needed. The reference method for the measurement of HbA_1c is an ion-exchange high-performance liquid chromatography (HPLC) according to the Diabetes Control and Complications Trial [7]. HbA_1c measurements can be adversely affected by conditions that disrupt the Hb composition [8]. Although there are many reports in the literature showing that Hb variant affects the accuracy of methods for HbA_1c measurement [8], [9]. HbA_1c values measured in patients with only HbA₂ and HbF abnormality were few [10], [11]. HbA₂ is co-eluted with Hb A0, therefore in the case of β-thalassemia carriers, the amount of HbA₂ may affect the elution profile of HbA_1c. Even though HbF fraction is eluted a little faster, it is difficult to separate from the HbA_1c peak in the case of highly enhanced HbF concentrations. Therefore, we investigated the potential effect of changes in HbA₂ and HbF on HbA_1c measurements by HPLC using the immunoturbidimetric assay as the comparative method.

Materials and methods

The study population consisted of 124 individuals admitted to the Biochemistry Laboratory of Zonguldak Bulent Ecevit University between 2013 and 2014 years. This study was approved by the Ethics Committee of the University of Zonguldak Bulent Ecevit University. The samples were selected from patients who showed a hemoglobin variant during the measurement of routine HbA_1c testing. All analyses were performed on a single whole-blood sample taken into an ethylenediaminetetraacetic acid (EDTA) tube. The samples were stored at 4 °C or −70 °C and analyzed within 5 days of the blood being drawn.

HbA_1c was assayed by ion-exchange HPLC (Spectra System, Thermo Scientific, Boston, MA, USA) using the commercially available kit ClinRep (Recipe Chemicals – Instruments GmbH, Munich, Germany) according to the manufacturer’s instructions. Calibration of instruments was done according to the manufacturer’s instructions. The method is linear over the whole range of physiologically relevant HbA_1c values (3–16% HbA_1c). The presence of abnormal patterns on the chromatograms were cross-checked using by ClinRep hemoglobin variant and β-thalassemia screening kit (Recipe Chemicals – Instruments GmbH, Munich, Germany) on same HPLC analyzer. Patients were identified according elevated HbA₂ (>3.5%) and/or HbF (>2%) and divided into three groups (Figure 1). HbA_1c values of groups were re-assayed by immunoturbidimetric method using manufacturer kit on an ADVIA 2400 analyzer (Siemens Healthcare, Erlangen, Germany). HbA_1c values are reported with both the National Glycohemoglobin Standardization Program (NGSP) (%) and International Federation of Clinical Chemistry (IFCC) (mmol/mol) units. In this article, HbA_1c results will be reported in percentage of HbA_1c units. Conversion of HbA_1c units is by the following equation [7]:

Figure 1:

Flowchart demonstrating the procedure of the study.

HbA1c(mmol/mol)=[HbA1c(%)−2.15]/0.092

The intra- and inter-assay coefficient of variation (CV) values in determination of HbA_1c by HPLC were 1–2% and 2–3%, respectively. The intra- and the inter-assay CVs for the immunoturbidimetric method were 2.2% and 2.6%, respectively.

Results

The present study was carried out among 124 subjects and categorized into three groups based on their hemoglobin variant analysis. The mean±standard deviation (SD) HbA₂ levels were significantly higher in the group 2 (4.8+0.5%) and 3 (4.7±0.5%) than the group 1 (2.1±0.5%) (p<0.016). HbF levels were significantly higher in the group 1 (4.2±2.3%) and 3 (4.3±2.2%) compared with the group 2 (1.0±0.5%) (p<0.016). The differences of the HbA_1c levels between HPLC and immunoturbidimetry for each group are shown in the Table 1. HbA_1c concentrations in all groups were showed lower results in HPLC methods compared to immunoturbidimetry (p<0.05).

With Passing and Bablok regression analysis, group 1 (Figure 2A) yielded the equation of y=1.11×(95% CI, 0.67–2.27)–2.64 (95% CI, −9.1 to −0.11); group 2 (Figure 2B) yielded the equation of y=1.46×(95% CI, 1.11–1.94)–3.31 (95% CI, −6.03 to −1.36); group 3 (Figure 2C) yielded the equation of y=1.62×(95% CI, 0.79–3.11)–4.18 (95% CI, −11.6 to −0.05). Calculation of Spearman’s correlation coefficient showed a positive correlation (r=0.49, p<0.0001) between the two methods in the group 2. A slight correlation (r=0.38, p=0.06) in the group 1 and no correlation (r=0.04, p=0.78) in the group 3 was observed between two methods.

Figure 2:

Spearman’s correlations coefficients of 2A, 2B and 2C, respectively (r=0.38, p=0.06; r=0.49, p<0.0001; r=0.04, p=0.78).

Passing-Bablok regression analysis of HbA_1c results obtained with the HPLC compared with immunoassay method in group 1 (A), 2 (B) and 3 (C).

Discussion

HbA_1c is glycated hemoglobin in which glucose is bound specifically to the N-terminal valine residue of one or both beta chains of hemoglobin via a nonenzymatic process. It reflects the mean glycemic level in the lifespan of the erythrocyte. The Diabetes Control and Complications Trial showed that a reduction of 1% HbA_1c corresponded to an approximate 30% reduction in the risk of diabetes complications [12].

Hemoglobin variants may interfere with the HbA_1c result in three ways; by decreasing the life span of red blood cells, by influencing the binding of glucose to Hb and by affecting the net charge of the hemoglobin [8], [13]. Ion-exchange HPLC separates Hb species based on charge differences, while immunoassay methods use antibodies that recognize the N-terminal glycated amino acids in the first four to 10 amino acids of the β-globin chain of the hemoglobin. In our study, we demonstrated that elevated HbA₂ and/or HbF can affect determination of HbA_1c by our HPLC method and might result in falsely lower HbA_1c concentrations. Analytic interference of immunoturbidimetric methods is extremely low in heterozygous Hb variant conditions in which the red blood cell lifespan may be normal [14]. However, they do not provide any information about the proportions of the different Hb fractions. Most ion exchange chromatography techniques separate HbF and Hb A₂ from HbA_1c and this can be a very precious information on medically silent patients [15]. Routine inspection of chromatograms allowed us to identify the presence of HbF and HbA₂, although it could not be presented in this paper.

In conclusion, chromatograms must be carefully examined to identify possible interference in HbA_1c measurement using HPLC. HbA_1c measurement by HPLC method interfering with elevated HbA₂ and HbF, especially HbF, should be verified by an immunoturbidimetric method.