Unexpected Detection of Abnormal Hemoglobin Variants During Routine HbA1c Analysis by HPLC Technique: Challenges and Opportunities in HbA1c Testing

Background: Glycated Hemoglobin A1c (HbA1c) assay is most widely used in diabetic patients for assessing long-term control of glycemia. The presence of hemoglobin variants may be an incidental finding and can interfere with HbA1c measurements. The aim of the present study is to investigate the prevalence and impact of interference of different abnormal hemoglobin variants on HbA1c measurements during routine HbA1c testing. Methods: A total of 12,092 HbA1c samples were collected from January to August 2018. HbA1c quantification was carried out on a Variant II Bio-Rad’s HPLC analyzer. Abnormal chromatograms were further analyzed using the extended-run high-pressure liquid chromatography (HPLC) analysis in the A2/F mode. Results: The samples were examined for presence of abnormal variants. Samples producing abnormal chromatograms were further analyzed in A2/F mode to characterize hemoglobin variants. Abnormal variants were identified in 126 (1%) samples, and 74 (0.59%) sickle cell traits (SCT) were the most common variant in our findings. Moreover, 30 (0.24%) cases were eluted in the variant window in A1c mode, which on further analysis were found to be Hb E Hb D traits. Furthermore, 3 (0.02%) cases were eluted at a RT 1 min as (unknown) and identified as Hb H. Also,19 (0.15%) samples were eluted in the P3 window at different retention times. Conclusion: Observing each chromatograph after the analysis can help us in identifying silent hemoglobin variants in routine HbA1c testing. Knowledge and awareness of common hemoglobin variants affecting measurement of HbA1c is imperative to avoid reporting of falsely low HbA1c values in diabetic population.