Identification of Clinically Relevant Hb Variants During HbA1c Testing Using the ARKRAY HA-8180V in Fast Mode: Reliable HbE Detection Based on Retention Time.

Abstract

Background: Hb variants are often incidentally detected during HbA1c testing and can cause interference. We evaluated the ability of the ARKRAY HA-8180V HPLC system operating in fast mode (FM) to identify Hb variants, particularly HbE, in samples submitted for HbA1c testing based on characteristic retention times (RTs).

Methods: Chromatograms of samples tested for HbA1c were analyzed; those showing predefined abnormal chromatographic patterns suggestive of Hb variants were further evaluated using capillary electrophoresis (CE) and β- and α-globin gene sequencing. RTs were recorded.

Results: Of 4,888 chromatograms analyzed, 588 (12.0%) were abnormal. Among these, 564 (95.9%) showed an abnormal peak at an RT of 23-25 s and were confirmed to contain HbE via CE. Identified Hb phenotypes included EA (N=526), compound heterozygotes [Constant Spring (CS) EA, N=2; CSEE, N=1], EE (N=27), EFA (N=3), and EF (N=5). Other variants included Hb CSA₂ABart'sH, A₂FA, A₂FABart's, and A₂F. Rare variants, Hb Hope (N=12) and Hb Korle-Bu (N=1), and two uncharacterized variants were also detected. Hb Hope interfered with HbA1c measurements, and all CSA₂ABart'sH samples produced falsely low HbA1c values. Artificially decreased HbA1c was detected in 21/27 (77.8%) HbEE samples. ARKRAY HA-8180V FM analysis yielded reproducible RTs for HbE, achieving 100% sensitivity, specificity, and accuracy in the validation cohort.

Conclusions: HbE and thalassemia variants can interfere with HbA1c quantification. ARKRAY HA-8180V FM chromatogram review allows reliable detection of HbE and prevents misinterpretation of HbA1c results.