| Summary: | Introduction: The visual detection of the early-eluting unknown peaks (UPs) in the chromatogram of the high-performance liquid chromatography (HPLC) is essential for its being supplementary to the diagnostic workup of different phenotypes of β-Thal, particularly in areas endemic to this disease. This study aimed to scrutinize and uncover the interaction of the level of UPs detected during HPLC analysis of hemoglobin (Hb) with the Hb-F, Hb-A2, Hb subunits levels, and β-Thalassemia (β-Thal) phenotypes. Materials and methods: A total of 161 respondents with β-Thal and 52 healthy blood donors were included in this study. HPLC analysis of Hb and quantification of the Hb subunits were performed using the VARIANT II β-Thalassemia Short Program and triple-quad mass spectrometry, respectively. The level of UPs was measured using the software ImageJ 1.53k. Results: The mean levels of UP2, UP3, and UP4 correlated positively with the quantities of Hb-A2 and Hb-F, mainly in Hb-E/β-Thal and β-Thal major groups. The mean level of UP4 showed a direct correlation with Hb-subunit gamma. Conclusion: The composition of the UP4 is gamma-globin in nature. Further exploration of other elements in these pre-integration UPs might uncover clinically valuable markers. Knowledge and awareness of how these UPs interact with the Hb variants in different β-Thal and their compositions, especially in cases of β-Thal major or Hb E/β-thal, might contribute toward revealing molecular mechanisms in β-Thal and reinforcing the hematopathologists’ interpretation of the result of HPLC analysis of Hb in diagnosing different β-Thal phenotypes. © 2024 Universiti Putra Malaysia Press. All rights reserved.
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