The peculiar feature of glycated hemoglobin (HbA1c) as a measure of chronic glycemic control makes it compelling to be used as an estimation of the average blood glucose over the past 120 days in monitoring as well as diagnosis of diabetes mellitus (DM).1 HbA1c is a hemoglobin (Hb) adduct resulting from irreversible glycation at either one or both of the N-terminal valines of beta chains.2 It can be quantified by various methods in which the principle of the measurement can be divided into three major categories which are depending upon charge difference, structural characteristics of glycogroups on Hb, and chemical reactivity. High-performance liquid chromatography (HPLC) is the most common method being used where HbA1c is abstracted from other Hb molecules in a column using charge difference.3,4 However, many factors such as Hb variants, carbamylated Hb, and hematological diseases interfere to varying degrees with these current analytical methods.2,5 Introduction of capillary electrophoresis (CE) method in HbA1c assessment has said to give a more accurate result in those patients with Hb variant.6 Here, we report two cases of Hb J with abnormally high HbA1c levels using HPLC and comparison with HbA1c value given by CE method.

Case report

Case one

A 26-year-old female Malay patient at 25 weeks gestation was first diagnosed with DM during antenatal booking at six weeks pregnancy. Her initial diabetic workup were: glycosuria 3+, random blood sugar 15.5 mmol/L, and HbA1c 18.5% (> 179 mmol/mol). Insulin treatment was commenced, and she was able to achieve good glycemic control with a fasting blood sugar (FBS) range of 4–6.1 mmol/L. However, her HbA1c result using HPLC showed a persistently high level (> 18.5%, > 179 mmol/mol). Other biochemical laboratory results were as follows (reference range in parentheses); sodium 135 mmol/L (135–145), potassium 4.1 mmol/L (3.5–5.0), urea 2.1 mmol/L (1.7–8.3), creatinine 59 umol/L (70–130), albumin 46 U/L (38–44), aspartate aminotransferase (AST) 18 U/L (5–34), and alanine aminotransferase (ALT) 11 U/L (< 34). Her full blood count (FBC) results were RBC 5.00 × 1012/L (3.52–5.16), Hb 14.8 g/dL (9.81–13.85), mean cell volume (MCV) 84.6 fL (77.5–94.5), and mean corpuscular Hb (MCH) 29.6 pg (24.8–31.2). This raised suspicion of the presence of a Hb variant. A full blood picture (FBP) and Hb analysis were sent. FBP showed normal RBC morphology and count with no features of thalassemia or hemoglobinopathy. Hb analysis by HPLC with beta-thalassemia short program revealed a 29% abnormal peak at P3 with a retention time (RT) of 1.47 min [Figure 1]. There was presence of additional band anodal to HbA on alkaline gel electrophoresis suggestive of Hb J. HbA1c test was rerun with a different platform using the CE principle. The presence of Hb J variant peak supports the finding of Hb analysis, and the result was good diabetic control with HbA1c of 6% (42 mmol/mol) with the use of CE.

Case two

A 62-year-old female Malay was diagnosed with type 2 DM two years prior and on an oral hypoglycemic agent. During the first year of treatment, the diabetes was not well controlled, with FBS ranging from 8.3–11.1 mmol/L, and her HbA1c was persistently > 18.5% (> 179 mmol/mol). Subsequently, the treatment was optimized and good glycemic control was achieved with average FBS of around 5–7 mmol/L. Despite good diabetic control based on FBS, the HbA1c remains high with a level of > 18.5% (> 179 mmol/mol). Other biochemical laboratory results were as follows: sodium 140 mmol/L, potassium 4.2 mmol/L, urea 3.9 mmol/L, creatinine 55 umol/L, albumin 43 U/L, AST 19 U/L, and ALT 21 U/L. Her FBC results were RBC 5.75 × 1012/L, Hb 16.3 g/dL, MCV 89.0 fL, and MCH 28.3 pg. Due to this condition, interference from Hb variant was taken into consideration. The FBP and Hb analysis results were similar to the previous patient; there was 29.8% abnormal peak at P3 with RT of 1.44 min [Figure 2]. Analysis of HbA1c using CE supports the presence of Hb J variant in this patient with a normalizing HbA1c level of 8.1% (65 mmol/mol).

Discussion

HbA1c is used to monitor and diagnose DM.1 HPLC is the most common method used where HbA1c is separated from other Hb molecules using charge differences.3,4 However, many factors such as Hb variants, carbamylated Hb, and hematological diseases interfere to varying degrees with this method.2,5 There are few causes that can produce significantly high HbA1c, such as hemoglobinopathies, iron or vitamin B12 deficiencies, and chronic renal failure (CRF).7 The cause of high HbA1c in both our cases was due to the presence of Hb J evidenced by Hb analysis and CE. CRF was excluded as the urea, electrolytes, and creatinine levels were normal. Iron or vitamin B12 deficiencies were also excluded since the FBC results showed no evidence of micro/macrocytic anemia. CE in HbA1c assessment has been said to give a more accurate result in patients with Hb variant.6 Hb variant is defined as Hb that has a substitution of single amino acid resulting in alteration in Hb structure and biochemical functions.8 In Southeast Asia, Hb E is the most prevalent accounting for 50%–60% near Thailand, Cambodia, and Laos, and 1%–8% are Hb constant spring.9 Hb J is an alpha-globin gene variant with rare incidence worldwide.10 Depending on its variant, Hb J has particular characteristics and functions. It can range from completely normal clinical features such as in Hb J Sardegna up to severe presentation like in heterozygous Hb J Capetown, which is associated with increased oxygen affinity and polycythemia. Other Hb J variants such as Hb J Bangkok and Baltimore are associated with sickle Hb.11 In both cases, the Hb J variant could not be determined as the Hb genotypes were not carried out due to the logistic problem. With the RT of 1.47 and 1.44 min, respectively, Hb J Bangkok and Hb J Singapore need to be considered. Being the less common occurrence of Hb disorders than Hb E trait (19.3%) and thalassemia,12 the impact of Hb J in HbA1c measurement interference may be undervalued. Incidental detection of Hb J during routine HbA1c test may add value in identifying this rare Hb variant, and other appropriate measures for glycemic control should be taken into consideration.

The mechanism of Hb variant interference in HbA1c measurement is method-dependent, and it can be classified into physiological or analytical factors. Physiologically, the Hb variant may disrupt the process of HbA1c formation, which will cause an underestimation of HbA1c.5 For instance, in the homozygous Hb variant, there will be no HbA1c formation because only the glycated form of the Hb variant can be found. Similarly, the heterozygous Hb variant will produce a lesser amount of HbA1c in addition to the glycated form of the Hb variant.9

In more detail, the Hb variant may interfere during the testing process by co-eluting with HbA1c peak if it carries a similar charge with HbA1c. This is valid, especially for the HLPC method.5 Depending on the mutation point in the variant and the assay used for measurement of HbA1c, each variant may cause a clinically misleading value of HbA1c, affecting the interpretation of the result.2,13 In the case of Hb J, there were few studies that reported lower HbA1c values in patients with this Hb variant6,11,14,15 in contrast to our patients who showed abnormally high HbA1c using HPLC method.

The repeated HbA1c using CE showed results within the normal range. The separation of HbA1c using CE is based on electrophoretic mobility and electroosmotic flow in an alkaline buffer with a specific pH. This segregation principle results in CE being more accurate in providing true HbA1c value and, at the same time, detection of Hb variant and other Hb adducts. CE can display the various peak of Hb components at different intervals and give better resolution than HPLC.16–19 This feature is being utilized to give more exact assessment of HbA1c in relation to glycemic control with minimization of interference from other factors. In addition, CE is also preferred over HPLC in terms of easy experimental setup, faster time for analysis, and requires very low reagent and sample as well as cost effective.20

Conclusion

Careful interpretation of HbA1c results is essential in populations with a relatively high prevalence of Hb variants, such as in Malaysia. Interference from a Hb variant should be suspicious when the HbA1c results do not correlate clinically or with blood glucose levels. This is crucial to prevent wrong diagnosis and mismanagement of the patients.

Disclosure

The authors declared no conflicts of interest. Both patients have given consent for their data to be published.

references

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