• 58-year-old Chinese female with clinically silent hemoglobinopathy

  • Normal glucose tolerance: fasting plasma glucose 5.6 mmol/L, plasma glucose 2 h after 75-g glucose loading 5.98 mmol/L

  • Abnormally low, implausible HbA1c results: 2.0%; 3.6% (16 mmol/mol) on retesting

  • Normal hemoglobin (Hb) level with increased turnover: Hb 136 g/L, reticulocyte percentage 6.53%

  • High-performance liquid chromatography (HPLC) indicated an abnormal peak

  • Heterozygous mutation in the HBB gene: c.242T>A, Leu81His

  • Same mutation and consistent phenotype in the daughter of the proband

  • HbA1c values estimated by HPLC are unreliable in patients with hemoglobinopathies, but visual inspection of the chromatogram can be used to identify hemoglobinopathies.

A 58-year-old woman presented with elevated fasting plasma glucose found in a medical check-up. She was asymptomatic, and clinical examination was normal apart from slight sclera jaundice. The 75-g oral glucose tolerance test showed fasting plasma glucose of 5.6 mmol/L and 2-h postprandial plasma glucose of 5.98 mmol/L. The HbA1c taken at the same visit was 2.0% (value unavailable in International Federation of Clinical Chemistry and Laboratory Medicine [IFCC] units) (assayed by the Tosoh G8 analyzer); on retesting, it was 3.6% (16 mmol/mol) (assayed by the Bio-Rad D-10 TM analyzer). The synchronous glycated albumin was 11.59%. Her other blood results were as follows: Hb 136 g/L, red blood cell count 4.35 × 1012/L, mean corpuscular volume 98.9 fL (normal range 82–100), mean corpuscular Hb 31.3 pg (normal range 27–34), and mean corpuscular Hb concentration 316 g/L (normal range 316–354). Further investigations showed elevated reticulocytes, elevated serum indirect bilirubin (indirect bilirubin 26.9 μmol/L, normal range <20; direct bilirubin 12.6 μmol/L, normal range <8.8; total bilirubin 39.5 μmol/L, normal range 5–28), normal transaminases, and negative fecal occult blood test. Haptoglobin was significantly decreased (<58.3 mg/L) and the methemoglobin reduction test was 7.7%, indicating hemolysis. The direct Coombs test was negative, and glucose-6-phosphate dehydrogenase activity was normal. Hb electrophoresis revealed two abnormal bands (assayed by Sebia CAPILLARYS 2), so hemoglobinopathy was suspected. Gene sequencing identified a heterozygous mutation (c.242T>A, Leu81His) in the HBB gene (1). During family screening, the daughter of the proband was found to have normal blood glucose, decreased HbA1c (3.9%, 19 mmol/mol) with abnormal bands on Hb electrophoresis tracing, and the same mutation (pedigree presented as Supplementary Fig. 1). Glycated albumin was used to monitor the patient’s glucose level afterward.

When reviewing the case, we further requested the HPLC chromatogram of this patient, in which an abnormal peak was presented (arrow, Fig. 1A) and was reported as the variant window. This indicated the capability of HPLC chromatograms in identifying unexpected variants. The HPLC chromatogram of the patient’s husband served as a normal control (Fig. 1B; both assayed by the Bio-Rad D-10 TM analyzer).

Figure 1

Chromatograms of HbA1c tests. A: HPLC chromatogram of the proband. Arrow indicates the abnormal peak. The HbA1c measured was 3.6% (16 mmol/mol). B: HPLC chromatogram of the husband of the proband as normal control. The HbA1c measured was 6.4% (47 mmol/mol).

Figure 1

Chromatograms of HbA1c tests. A: HPLC chromatogram of the proband. Arrow indicates the abnormal peak. The HbA1c measured was 3.6% (16 mmol/mol). B: HPLC chromatogram of the husband of the proband as normal control. The HbA1c measured was 6.4% (47 mmol/mol).

Close modal

HbA1c is widely used as an independent diagnostic criterion for diabetes and as a monitor for glycemic control (24). However, under several circumstances, HbA1c by itself does not accurately reflect the plasma glucose level (5,6). International guidelines have called clinicians’ attention to several comorbid conditions in which HbA1c is unreliable, such as hemoglobinopathy and hemolytic anemia (2). In the presented case, an increased erythrocyte turnover and shortened red blood cell life span resulted in the abnormally decreased HbA1c reading. The majority of assay methods certified by the NGSP (formerly, National Glycohemoglobin Standardization Program) are not affected by common Hb traits (7), but if an Hb variant is detected, additional workup to further characterize the Hb variant may be necessary, and results should be correlated with complete blood count data and with clinical findings.

HPLC is one of the common assays for HbA1c measurement (8), and abnormal HPLC chromatograms may inform clinicians and laboratory staff of potential interferences. In the presented case, the HPLC chromatogram was requested post hoc because of the extremely abnormal HbA1c value, but it could easily be overlooked if the hemoglobin variant caused only mild interference. Although the necessity of presenting chromatograms routinely to clinicians needs further discussion, it is acknowledged that when measuring HbA1c by a chromatographic technique, visual inspection of chromatograms prior to reporting of HbA1c results is warranted, especially when there is discordance between HbA1c and other clinical data.

Acknowledgments. The authors thank Haoming Tian (Department of Endocrinology and Metabolism, West China Hospital, Sichuan University, Chengdu, China), Ewan Pearson (Division of Cardiovascular and Diabetes Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, U.K.), and Hui Yang (Department of Laboratory Medicine, West China Second University Hospital, Sichuan University, Chengdu, China) for valuable comments on the manuscript. The authors thank Dr. Qian Niu (Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, China) for help with interpreting the laboratory results.

Funding. This research received no specific funding from any bodies in the public, commercial, or not-for-profit sectors. S.L. was supported by grants from the National Natural Science Foundation of China (grant numbers 81400811 and 21534008), the National Basic Research Program of China (grant number 2015CB942800), the Scientific Research Project of Health and Family Planning Commission of Sichuan Province (grant numbers 130029, 150149, 17PJ063, and 17PJ445), the Cholesterol Fund by China Cardiovascular Foundation and China Heart House, and the International Visiting Program for Excellent Young Scholars of Sichuan University.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. Q.L. and S.L. wrote the manuscript. Y.X. interpreted the laboratory results. A.D.S. critically reviewed the manuscript. S.L. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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Supplementary data