False hemoglobin A_1c value as a result of compound heterozygotes of hemoglobin E and hemoglobin New York

Abstract

The presence of hemoglobin (Hb) variants might interfere with some glycated hemoglobin (HbA_1c) measurements. There have been a few reports of compound Hb variants affecting HbA_1c testing. Here, we report a case of the coinheritance of two Hb variants in the β‐globin gene. High‐performance liquid chromatography with the Hb program showed a high HbA₂ level. Similarly, an E‐window peak was separated on the high‐performance liquid chromatography with a glycated Hb program. However, capillary electrophoresis showed two abnormal peaks and no HbA peak. Sanger sequencing confirmed the presence of Hb New York and HbE. This is the first report of a compound heterozygote for HbE and Hb New York. The double heterozygote caused erroneous results for HbA_1c on high‐performance liquid chromatography and enzyme assay.

In this study, we report a case of the double hemoglobin (Hb) variants heterozygotes in the β‐globin gene, the first reported compound heterozygote for HbE and Hb New York. High‐performance liquid chromatography only showed HbE whether by glycated Hb program or Hb program, but capillary electrophoresis detected HbE and Hb New York. The double heterozygote caused erroneous results for HbA_1c on high‐performance liquid chromatography and enzyme assay.

INTRODUCTION

Glycated hemoglobin (HbA_1c) is widely considered the most reliable marker for diagnosing diabetes and monitoring glucose control ¹ . There are many reports that the presence of a hemoglobin (Hb) variant might interfere with the measurement of HbA_1c results ² , ³ , ⁴ . Heterozygous HbE, the common β‐globin variant, possesses an influence on the HbA_1c value ⁵ , ⁶ . The effect of heterozygous Hb New York on HbA_1c value is observed in different assays ⁷ . However, little information is available on the determination of HbA_1c affected by compound Hb variants in the β‐globin gene. Here, we report that the judgment of HbA_1c might be affected by Hb New York and HbE using high‐performance liquid chromatography (HPLC) and enzyme assay.

CASE REPORT

A 26‐year‐old woman in the first trimester of pregnancy presented to the Women and Children's Care Hospital of Xing'an County, Guilin, Guangxi, China, for a pregnancy examination. Written informed consent was obtained from the patient, and samples were collected. The hematological parameters showed the following: Hb level 11.8 g/dL, red cell count 4.76 × 10¹²/L, mean corpuscular volume 72.7 fL and mean corpuscular Hb 24.8 pg (Sysmex XN 550; Sysmex Corporation, Kobe, Japan). Her fasting blood glucose level was 5.0 mmol/L (AU680; Beckman Coulter, Brea, CA, USA). HbA_1c measurement was carried out using HPLC (D100; Bio‐Rad, Hercules, CA, USA) and enzyme assay (AU680; Beckman Coulter).

According to local policy, a pregnant woman can receive free screening tests. Thalassemia screening was carried out using HPLC (VARIANT II™; Bio‐Rad). Capillary electrophoresis (CE; CapillaryS2 Flex Piercing; Sebia, Lisses, Paris, France) was used to screen the potential Hb variants compared with HPLC. Routine genetic analysis was used to rule out the presence of α‐thalassemia (Yishengtang Biotech, Shenzhen, China). Finally, Sanger sequencing was carried out with a 3,500 XL sequencer (Applied Biosystems, Foster City, CA, USA).

D100 showed HbF 1.24%, HbA_1c 5.15%, HbA0 54.89% and E‐window peak 31.31%, which suggested the presence of HbE (Figure 1a). In view of the aberrant peak, the HbA_1c value was then determined by enzyme assay, and the HbA_1c value was 3.88%. Hb analysis showed a high HbA₂ value (28.9%) by VARIANT II, suggesting a potential Hb variant co‐eluting with HbA₂ (Figure 1b). No mutations were detected by routine genetic analysis. Sanger sequencing detected a single‐nucleotide substitution (G>A) at codon 26 in the HBB gene, corresponding to HbE (β26 [B8] Glu >Lys, HBB:c. 79 G>A; Figure 2a). Accidentally, we found that there was another mutation (T>A) at codon 113 in the HBB gene, which corresponded to Hb New York (β113 [G15] Val >Glu, HBB:c. 341 T>A; Figure 2b). Subsequently, CE was confirmed, and presented values of HbE 26.9%, Hb New York 71.3% and HbA₂ 1.8% (Figure 3a).

Figure 1.

(a) Glycated hemoglobin (HbA_1c) and (b) hemoglobin (Hb) analysis for the patient with compound heterozygous HbE and Hb New York by high‐performance liquid chromatography.

Figure 2.

Sanger sequencing showed compound heterozygous (a) hemoglobin (Hb)E and (b) Hb New York in the patient, and (c) heterozygous HbE in her mother.

Figure 3.

Results of hemoglobin (Hb) analysis by capillary electrophoresis for (a) the patient and (b) her mother.

To investigate more information, we carried out a pedigree study. Unfortunately, the patient had no brothers or sisters, and her father died. Her mother's (age 51 years) related measurement results were as follows: Hb 119 g/dL, mean corpuscular volume 80.6 fL, mean corpuscular Hb 26.2 pg; HbA 70.2%, HbA₂ 3.6%, HbE 26.2% (by CE; Figure 3b); HbA_1c 5.4% by HPLC and HbA_1c 5.2% by enzyme assay; CD26 heterozygous mutation in the β‐globin gene (Figure 2c).

DISCUSSION

In the present study, we detected compound heterozygous HbE and Hb New York in a patient with a false HbA_1c value, as measured by HPLC and enzyme assay. Interestingly, HPLC could not detect Hb New York in either glycation analysis or Hb analysis mode (Figure 1) ⁸ . If a CE re‐examination was not carried out, the patient was likely to be considered heterozygous for HbE. The HbA_1c value by enzyme assay was 3.88%, whereas the HPLC was 5.15%. Based on the normal fasting blood glucose of the patient, the HbA_1c value by HPLC was considered reasonable. However, Sanger sequencing showed two variant mutations on the β‐globin chain, which suggested that the measurement HbA_1c results by HPLC and enzyme assay were incorrect.

As far as we know, enzyme assay is often considered a reference method when the Hb variants are presented. The patient was compound heterozygous HbE and Hb New York in the present study. It suggested no normal β‐chain generation, and HbA could not be synthesized. The CE confirmed this speculation, and no HbA was detected. However, the present study showed a low HbA_1c value (3.88%) by enzyme assay. Obviously, this was a false HbA_1c value. Therefore, assays, such as HPLC and CE, are recommended to screen Hb variants or thalassemia, and avoid misdiagnosis of diabetes. Enzyme assay might be considered when the presentence of the Hb variant interferes with HbA_1c measurement. It was observed that the result of HbA_1c using HPLC was different from using the enzyme assay from the patient's mother. When double heterozygotes or homozygotes for β‐globin chain variants are encountered, HbA_1c cannot be used as a detection indicator, regardless of the methods used.

In conclusion, this is the first report of the compound heterozygous HbE and Hb New York with false HbA_1c measurement. It should prompt clinicians to be careful about Hb variants when the HbA_1c value is outside the laboratory's reference range and not consistent with the clinical picture. Because Hb variants can lead to abnormal HbA_1c values through various mechanisms, no single method can accurately measure HbA_1c. As in the present study, both HPLC and enzyme assay received false HbA_1c results from the patient with compound Hb variants. Therefore, the assessment of glycemic control should be changed to use other indicators.

DISCLOSURE

The authors declare no conflict of interest.

Approval of the research protocol: N/A.

Informed consent: Informed consent was obtained from participants in this study.

Registry and registration no. of the study/trial: N/A.

Animal studies: N/A.