Hemoglobin variants uncovered during routine HbA1c testing: Diagnostic pitfalls and clinical significance

Introduction

Hemoglobin A1c (HbA1c) serves as a reliable indicator of average plasma glucose levels over the preceding 8 to 12 weeks. An HbA1c threshold of 6.5% or higher is diagnostic for diabetes mellitus (DM), as recommended by international guidelines [1] . The National Glycohemoglobin Standardization Program (NGSP) has validated several methodologies for HbA1c measurement, including iso-electric focusing, ion-exchange high-performance liquid chromatography (HPLC), boronate-affinity chromatography, immunoassay, enzymatic assay, and capillary electrophoresis [2] . Among these, ion-exchange HPLC and capillary electrophoresis offer the advantage of generating chromatograms with distinct hemoglobin fraction peaks based on retention times.This graphical output allows not only HbA1c quantification but also the incidental detection of hemoglobin variants, which may have significant clinical implications. Many hemoglobinopathies are geographically linked, with higher prevalence in Southeast Asia, the Indian subcontinent, the Mediterranean, and parts of Africa. In India, hemoglobin variants such as HbE, HbS, HbD, and high HbF are frequently encountered, especially in specific ethnic groups and regions [15,16] . Traditionally, these disorders are detected through targeted screening using hematological indices, HPLC or electrophoresis, and confirmed by molecular methods. However, with the increasing use of automated HPLC systems for HbA1c measurement in diabetes care, hemoglobin variants may be incidentally uncovered in patients undergoing routine monitoring [5,6] .

Detecting these variants during HbA1c testing provides an opportunity for earlier diagnosis, which could otherwise be missed in asymptomatic carriers. The incidental detection of hemoglobin variants during HbA1c testing poses both diagnostic and clinical challenges. Certain variants may interfere with HbA1c quantification, leading to falsely low or high values depending on the analytical method used [8] . Misinterpretation of such results can affect diabetes diagnosis and management. Therefore, laboratories must carefully review chromatograms and be equipped to identify variant patterns. This awareness is especially important in multiethnic populations and regions with evolving epidemiology of hemoglobinopathies due to migration, where unexpected variants may emerge outside their traditional geographic zones [9][10][11] . Detection of such variants during routine HbA1c testing presents an opportunity for early diagnosis of hemoglobinopathies, particularly in regions with high prevalence of genetic hemoglobin disorders.We conducted a study to determine the incidence and distribution of hemoglobin variants detected during routine HbA1c testing in a tertiary care hospital in South India, and to classify these variants using chromatographic parameters.

Aim

To determine the incidence and spectrum of hemoglobin variants detected incidentally during routine HbA1c testing using ion-exchange HPLC in a tertiary care center in South India. Objectives 1. To classify hemoglobin variants based on chromatographic retention times and peak patterns observed during HbA1c testing. 2. To evaluate the potential impact of hemoglobin variants on the reliability of HbA1c measurement and its implications for clinical management.

Methods

We performed a prospective observational study analyzing chromatograms from all HbA1c tests conducted over a continuous 24-months period (July 2018-June 2020) at a Department of Biochemistry and Department of General Medicine, in a tertiary care center in South India. The study included 10,200 unique HbA1c samples, excluding followup and duplicate samples from previously tested individuals.

HbA1c quantification was carried out using ion-exchange HPLC on the Bio-Rad D10 dual HbA2/F/A1c platform (Bio-Rad Laboratories Inc., California, USA), which has a reportable HbA1c range of 3.7-18.4%. This analyzer offers two operational modes:  A short program (3-minute HbA1c mode) for routine HbA1c measurement.  An extended program (6.5-minute HbA2/F mode) for quantifying HbA2 and HbF and detecting hemoglobin variants.

All HbA1c chromatograms obtained from the 3-minute runs were prospectively reviewed. Samples displaying unidentified peaks exceeding 6% of the total area were subsequently reanalyzed in the extended 6.5-minute mode to classify the hemoglobin variant, based on retention time and peak characteristics. Variant identification was crossreferenced with known retention times from the manufacturer's guidelines.

For patients with detected variants, HbA1c results were reported only after confirming that the variant did not interfere with HbA1c quantification. As per manufacturer's instructions, HbA1c values were not reported in cases with HbF or P3 peaks exceeding 10%, or in homozygous or compound heterozygous variants. Additionally, we compared HbA1c values obtained from the short (HbA1c) and extended (HbA2/F) modes to assess analytical agreement. This comparison was evaluated using intra-class correlation coefficient (ICC).

Results

Table The analysis of 85 hemoglobin variant cases among 10,200 HbA1c samples revealed that HbD heterozygous was the most frequently detected variant (27.1%), followed closely by HbE heterozygous (25.9%) and HbS heterozygous (22.4%). Gender distribution across variants showed a male predominance, particularly in HbD (60.9% male) and HbE (54.5% male), while compound heterozygosity for HbD-β thalassemia occurred exclusively in a female patient. The age distribution indicated that most variants were identified in middle-aged adults, with mean ages ranging from 46.2 to 52.5 years, except for compound heterozygous cases presenting at 35 years. HbA1c levels varied substantially across variants, with HbD heterozygous showing the widest range (4.5% to 13.8%, mean 7.8%), suggesting possible assay interference or variable glycemic control in this group.

Overall, these findings highlight the importance of recognizing variant hemoglobins during HbA1c interpretation, given their differential prevalence across age, sex, and glycemic ranges.

Discussion

Hemoglobinopathies are highly prevalent across Southeast Asia, Mediterranean regions, and Africa [15] . In India, the most frequently encountered hemoglobin disorders include beta-thalassemia, HbE, high HbF (suggestive of hereditary persistence of fetal hemoglobin [HPFH] or delta-beta thalassemia trait), HbS, and HbD. Traditionally, these conditions are identified through screening initiatives in atrisk populations, utilizing red blood cell indices, highperformance liquid chromatography (HPLC), or electrophoretic techniques, with confirmatory diagnosis via molecular genetic analysis [15,16] . Interestingly, hemoglobin variants can also be incidentally detected during routine HbA1c testing in diabetic individuals [5,6] . Instruments like the Bio-Rad D10 HPLC analyzer automatically generate chromatograms for every HbA1c assay, necessitating careful review to identify variant windows before releasing HbA1c results [6] . While several case reports have documented incidental detection of hemoglobin variants during HbA1c testing in India-including cases of HbQ (7), HPFH [8] , HbD Iran with beta-thalassemia [9] , HbJ Meerut [10] , HbE disease [11] , Hb Hope [12,13] , and delta-beta thalassemia trait [14] -our study represents one of the largest single-center cohorts, analyzing 10,200 HbA1c samples and detecting hemoglobin variants in 85 patients (0.83%). The predominant variants identified were HbD heterozygous (27.1%), HbE heterozygous (25.9%), and HbS heterozygous (22.4%), aligning with the known distribution of hemoglobinopathies across India [1518] . High HbF was the fourth most common variant (12.9%), consistent with patterns reported in northern and eastern Indian populations [19,20] . An interesting observation in our study was the presence of HbE heterozygosity (25.9%) in Karnataka, a state where earlier studies reported negligible HbE prevalence [16] . This apparent shift in epidemiological distribution may be attributable to increased interstate migration over the past two decades, potentially introducing hemoglobinopathies into regions previously considered low-prevalence zones [15] . Moreover, more than half of the patients with HbS trait in our series were of non-Indian origin, reflecting the influence of medical tourism from African and West Asian countries, further diversifying the local hemoglobinopathy spectrum. Despite beta-thalassemia trait being the most prevalent hemoglobinopathy in India [20] , it was not detected in our cohort due to the limitations of the HbA1c testing mode, which cannot differentiate beta-thalassemia trait in the chromatogram. Clinicians should remain vigilant for betathalassemia trait in patients presenting with a discordantly low HbA1c despite high fasting or postprandial glucose values. These individuals often exhibit a normal HbA1c chromatogram but show microcytosis (low MCV), a Mentzer's index <13, and normal iron studies-warranting further diagnostic evaluation.

A key clinical implication of our findings is the unreliability of HbA1c measurements in patients with homozygous or compound heterozygous hemoglobin variants, attributable to markedly reduced or absent HbA levels, which compromises glycation-based assays. Using HbA1c in such individuals risks misdiagnosis and mismanagement of diabetes [21] . Therefore, for patients with homozygous or compound heterozygous variants, alternative glycemic monitoring methods like fructosamine, glycated serum albumin, or capillary glucose monitoring are recommended. In contrast, in heterozygous states, assay interference depends on the specific analytical method. The Bio-Rad D10, as used in our study, has been shown not to interfere with HbA1c quantification in heterozygous states for most variants, except in cases with high HbF or where the P3 peak exceeds 10% [21] . Even so, it is critical to inform patients about their carrier status, as genetic counseling can help prevent homozygous or compound heterozygous outcomes in future generations. Unfortunately, due to resource limitations, confirmatory genetic testing was not performed in our study. In summary, our findings underscore the added diagnostic value of HbA1c testing as an opportunistic screening tool for hemoglobin variants, highlighting the need for routine chromatogram review to ensure that hemoglobinopathies are not overlooked, and that reported HbA1c values are valid and clinically actionable.

Conclusion

This study highlights the added diagnostic value of reviewing chromatograms during routine HbA1c testing for the incidental detection of hemoglobin variants. Among 10,200 samples, hemoglobin variants were identified in 0.83% of cases, with HbD, HbE, and HbS heterozygous states being the most prevalent. The detection of such variants has important clinical implications, as some may interfere with HbA1c measurement, leading to inaccurate assessment of glycemic control and potential mismanagement of diabetes. Laboratories should implement protocols for variant detection, interpretation, and reporting to ensure accurate clinical decisions. Furthermore, identifying hemoglobin variants provides an opportunity for early diagnosis, genetic counseling, and prevention of homozygous or compound heterozygous states in future generations. Routine chromatogram review should therefore be integrated as a standard practice in laboratories using HPLC-based HbA1c analysis, particularly in regions with a high prevalence of hemoglobinopathies.

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