Interaction of Hemoglobin-F, Hemoglobin-A2, Hemoglobin Subunits and Clinical Phenotypes of β-Thalassemia with the Levels of Early-eluting Unknown Peaks during High-performance Liquid Chromatography Analysis of Hemoglobin
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Abstract
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.
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Weatherall D.J., Clegg J.B. Inherited hemoglobin disorders: an increasing global health problem. Bull World Health Organ. 2001;79:704-12.PMID: 11545326 PMCID: PMC2566499.
Ou C.N., Rognerud C.L. Diagnosis of hemoglobinopathies: electrophoresis vs. HPLC. Clinica chimica acta. 2001;313:187-94.DOI: 10.1016/s0009-8981(01)00672-6
Pullon BM, Brennan SO. Two Familial Cases of Hb Tyne Confirm Instability as Cause of Low Expression. Thalassemia Reports. 2017; 7(1):6504. https://doi.org/10.4081/thal.2017.6504
Joutovsky A, Hadzi-Nesic J, Nardi M.A. HUPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: a study of 60000 samples in a clinical diagnostic laboratory. Clin Chem 2004;50:1736-1747. DOI: 10.1373/clinchem.2004.034991
Mohapatra M.K., Bariha, P. K., Tudu, K., Jajodia, N. K.. Examination of the primary fraction of hemoglobin in individuals having sickle cell disease using high-performance liquid chromatography. Asian Journal of Medical Sciences, 2022:13:68–71. https://doi.org/10.3126/ajms.v13i6.43634
Samperi U.P., Mancuso G.R., Dibenedetto S.P., Cataldo A. D.I., Ragusa R., Schilirò G. High performance liquid chromatography (HPLC): a simple method to quantify HbC, O-Arab, Agenogi, and F. Clin Lab Haematol 1991;13:169–75. DOI: 10.1111/j.1365-2257.1991.tb00266.x
Munkongdee T, Chen P,Winichagoon P, Fucharoen S andPaiboonsukwong K. Updatein Laboratory Diagnosis of Thalassemia. Front. Mol. Biosci.2020;7:74.doi: 10.3389/fmolb.2020.00074.
Clarke G.M, Higgins T.N. Laboratory Investigation of Hemoglobinopathies and Thalassemias: Review and Update. Clinical Chemistry 2000;46:1284–90. doi.org/10.1093/clinchem/46.8.1284.
Bain B.J., Wild B, Stephens A.D., Phelan L. (2010) Variant Haemoglobins: A guide to Interpretation ISBN-13: 978-1405167154. page: 446.
Gupta P.K., Kumar S, Jaiprakash M. Cation Exchange High performance Liquid Chromatography for Diagnosis of Haemoglobinopathies. Med J Armed Forces India. 2009;65:33-37. doi: 10.5662/wjm.v6.i1.20
Van Kirk R., Sandhaus L.M., Hoyer J.D. The detection and diagnosis of hemoglobin A2’ by high-performance liquid chromatography. Am J Clin Pathol 2005;123:657-661.PMID: 15981805.
Ercan Karadağ M, Akbulut E, Avcı E, Fırat Oğuz E, Kader S, Abuşoğlu G, Serdar M, Yılmaz F. Evaluation of four different HPLC devices for hemoglobinopathy screening. Turkish Journal of Biochemistry. 2021;46: 39-44. doi.org/10.1515/tjb-2019-0484.
Nair S.B. Potential Pitfalls in Using HPLC and its Interpretation in Diagnosing HbS. J Rare Dis Res Treat. 2018; 3: 9-12. doi.org/10.29245/2572-9411/2018/3.1161.
Warghade S, Britto J, Haryan R, Dalvi T, Bendre R, Chheda P, et al. Prevalence of hemoglobin variants and hemoglobinopathies using cation-exchange high-performance liquid chromatography in central reference laboratory of India: A report of 65779 cases. J Lab Physicians 2018;10:73-9.doi: 10.4103/JLP.JLP_57_17.
Abdullah U.Y.H, Al-Attraqchi A.G.F. Reasons of unknown HPLC peaks during hemoglobin analysis for patients with beta thalassemias. J Hematol Thrombo Dis 2014;2:5. 2014;2:5. doi. org/10.4172/2329-8790.1000160
Abdullah U.Y.H, Ibrahim H.M, Mahmud N, et al. Genotype-Phenotype Correlation of β-Thalassemia in Malaysian Population: Toward Effective Genetic Counseling. Hemoglobin 2019. DOI: 10.1080/03630269.2020.1781652.
Papadea C, Cate J.C. Identification and quantification of hemoglobins A, F, S, and C by automated chromatography. Clin Chem 1996;42:57-63.PMID: 8565233.
Trent R.J.A. Diagnosis of the Haemoglobinopathies. Clin Biochem Rev 2006;27:27-38.PMID: 16886045; PMCID: PMC1390791.
Abdullah U.Y.H, Ibrahim H.M, Jassim H.M, et al. Relative proteome quantification of alpha, beta, gamma, and delta globin chains in early eluting peaks of Bio-Rad variant II® CE-HPLC of hemoglobin from healthy and beta-thalassemia subjects in Malaysia. Biochemistry and Biophysics Reports 18 (2019) 100635. doi.org/10.1016/j.bbrep.2019.100635.
Abdullah U.Y.H, Ibrahim H.M, Mahmud N, et al. Quadrupole Time-of-Flight Mass Spectrometric Identification of Hemoglobin Subunits α, β, γ and δ in Unknown Peaks of High Performance Liquid Chromatography of Hemoglobin in β-Thalassemias, Hemoglobin 2019, DOI: 10.1080/03630269.2019.1632893.
Abdullah U.Y.H, Al-Attraqchi A.G.F, Ibrahim H.M, et al. The Free Alpha-Hemoglobin: A Promising Biomarker for β-Thalassemia. J Mol Biomark Diagn.2014;5:5. doi.org/10.4172/2155- 9929.1000197.
Kittivorapart J, Crew V.K., Wilson M.C., et al. Quantitative proteomics of plasma vesicles identify novel biomarkers for hemoglobin E/ β-thalassemic patients. Blood Advances 2018;2:10. doi: 10.1182/bloodadvances.2017011726.
Kheansaard W, Phongpao K, Paiboonsukwong K, Pattanapanyasat K, Chaichompoo P, Svasti S. Microparticles from β-thalassaemia/HbE patients induce endothelial cell dysfunction. Sci Rep 2018;8:13033. DOI:10.1038/s41598-018-31386-6.
Lin Y., Agarwal A.M., Anderson L.C., Marshall A.G. Discovery of a biomarker for β-Thalassemia by HPLC-MS and improvement from Proton Transfer Reaction – Parallel Ion Parking. Journal of Mass Spectrometry and Advances in the Clinical Lab 2023;28:20–26. doi.org/10.1016/j.jmsacl.2023.01.004.
Weatherall D.J., Clegg J.B. Inherited hemoglobin disorders: an increasing global health problem. Bull World Health Organ. 2001;79:704-12.PMID: 11545326 PMCID: PMC2566499.
Ou C.N., Rognerud C.L. Diagnosis of hemoglobinopathies: electrophoresis vs. HPLC. Clinica chimica acta. 2001;313:187-94.DOI: 10.1016/s0009-8981(01)00672-6
Pullon BM, Brennan SO. Two Familial Cases of Hb Tyne Confirm Instability as Cause of Low Expression. Thalassemia Reports. 2017; 7(1):6504. https://doi.org/10.4081/thal.2017.6504
Joutovsky A, Hadzi-Nesic J, Nardi M.A. HUPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: a study of 60000 samples in a clinical diagnostic laboratory.Clin Chem 2004;50:1736-1747. DOI: 10.1373/clinchem.2004.034991
Mohapatra M.K., Bariha, P. K., Tudu K., Jajodia, N. K.. Examination of the primary fraction of hemoglobin in individuals having sickle cell disease using high-performance liquid chromatography. Asian Journal of Medical Sciences, 2022:13:68–71. https://doi.org/10.3126/ajms.v13i6.43634.
Samperi U.P., Mancuso G.R., Dibenedetto S.P., Cataldo A. D.I., Ragusa R., Schilirò G. High performance liquid chromatography (HPLC): a simple method to quantify HbC, O-Arab, Agenogi, and F. Clin Lab Haematol 1991;13:169–75. DOI: 10.1111/j.1365-2257.1991.tb00266.x
Munkongdee T, Chen P,Winichagoon P, Fucharoen S andPaiboonsukwong K. Updatein Laboratory Diagnosis of Thalassemia. Front. Mol. Biosci.2020;7:74.doi: 10.3389/fmolb.2020.00074.
Clarke G.M, Higgins T.N. Laboratory Investigation of Hemoglobinopathies and Thalassemias: Review and Update. Clinical Chemistry 2000;46:1284–90. doi.org/10.1093/clinchem/46.8.1284.
Bain B.J., Wild B, Stephens A.D., Phelan L. (2010) Variant Haemoglobins: A guide to Interpretation ISBN-13: 978-1405167154. page: 446.
Gupta P.K., Kumar S, Jaiprakash M. Cation Exchange High performance Liquid Chromatography for Diagnosis of Haemoglobinopathies. Med J Armed Forces India. 2009;65:33-37. doi: 10.5662/wjm.v6.i1.20
Van Kirk R., Sandhaus L.M., Hoyer J.D. The detection and diagnosis of hemoglobin A2’ by high-performance liquid chromatography. Am J Clin Pathol 2005;123:657-661.PMID: 15981805.
Ercan Karadağ M, Akbulut E, Avcı E, Fırat Oğuz E, Kader S, Abuşoğlu G, Serdar M, Yılmaz F. Evaluation of four different HPLC devices for hemoglobinopathy screening. Turkish Journal of Biochemistry. 2021;46: 39-44. doi.org/10.1515/tjb-2019-0484.
Nair S.B. Potential Pitfalls in Using HPLC and its Interpretation in Diagnosing HbS. J Rare Dis Res Treat. 2018; 3: 9-12. doi.org/10.29245/2572-9411/2018/3.1161.
Warghade S, Britto J, Haryan R, Dalvi T, Bendre R, Chheda P, et al. Prevalence of hemoglobin variants and hemoglobinopathies using cation-exchange high-performance liquid chromatography in central reference laboratory of India: A report of 65779 cases. J Lab Physicians 2018;10:73-9.doi: 10.4103/JLP.JLP_57_17.
Abdullah U.Y.H, Al-Attraqchi A.G.F. Reasons of unknown HPLC peaks during hemoglobin analysis for patients with beta thalassemias. J Hematol Thrombo Dis 2014;2:5. 2014;2:5. doi. org/10.4172/2329-8790.1000160
Abdullah U.Y.H, Ibrahim H.M, Mahmud N, et al. Genotype-Phenotype Correlation of β-Thalassemia in Malaysian Population: Toward Effective Genetic Counseling. Hemoglobin 2019. DOI: 10.1080/03630269.2020.1781652.
Papadea C, Cate J.C. Identification and quantification of hemoglobins A, F, S, and C by automated chromatography. Clin Chem 1996;42:57-63.PMID: 8565233.
Trent R.J.A. Diagnosis of the Haemoglobinopathies. Clin Biochem Rev 2006;27:27-38.PMID: 16886045; PMCID: PMC1390791.
Abdullah U.Y.H, Ibrahim H.M, Jassim H.M, et al. Relative proteome quantification of alpha, beta, gamma, and delta globin chains in early eluting peaks of Bio-Rad variant II® CE-HPLC of hemoglobin from healthy and beta-thalassemia subjects in Malaysia. Biochemistry and Biophysics Reports 18 (2019) 100635. doi.org/10.1016/j.bbrep.2019.100635.
Abdullah U.Y.H, Ibrahim H.M, Mahmud N, et al. Quadrupole Time-of-Flight Mass Spectrometric Identification of Hemoglobin Subunits α, β, γ and δ in Unknown Peaks of High Performance Liquid Chromatography of Hemoglobin in β-Thalassemias, Hemoglobin 2019, DOI: 10.1080/03630269.2019.1632893.
Abdullah U.Y.H, Al-Attraqchi A.G.F, Ibrahim H.M, et al. The Free Alpha-Hemoglobin: A Promising Biomarker for β-Thalassemia. J Mol Biomark Diagn.2014;5:5. doi.org/10.4172/2155- 9929.1000197.
Kittivorapart J, Crew V.K., Wilson M.C., et al. Quantitative proteomics of plasma vesicles identify novel biomarkers for hemoglobin E/ β-thalassemic patients. Blood Advances 2018;2:10. doi: 10.1182/bloodadvances.2017011726.
Kheansaard W, Phongpao K, Paiboonsukwong K, Pattanapanyasat K, Chaichompoo P, Svasti S. Microparticles from β-thalassaemia/HbE patients induce endothelial cell dysfunction. Sci Rep 2018;8:13033. DOI:10.1038/s41598-018-31386-6.
Lin Y., Agarwal A.M., Anderson L.C., Marshall A.G. Discovery of a biomarker for β-Thalassemia by HPLC-MS and improvement from Proton Transfer Reaction – Parallel Ion Parking. Journal of Mass Spectrometry and Advances in the Clinical Lab 2023;28:20–26. doi.org/10.1016/j.jmsacl.2023.01.004.