Abstract
Objectives
Traditional methods for β-thalassemia screening usually rely on the structural integrity of hemoglobin (Hb), which can be affected by the hemolysis of red blood cells and Hb degradation. Here, we aim to develop a reliable and high throughput method for rapid detection of β-thalassemia using dried blood spots (DBS).
Methods
Hb components were extracted from a disc (3.2 mm diameter) punched from the DBS samples and digested by trypsin to produce a series of Hb-specific peptides. An analytical system combining high-resolution mass spectrometry and high-performance liquid chromatography was used for biomarker selection. The selected marker peptides were used to calculate delta/beta (δ/β) and beta-mutated/beta (βM/β) globin ratios for disease evaluation.
Results
Totally, 699 patients and 629 normal individuals, aged 3 days to 89 years, were recruited for method construction. Method assessment showed both the inter-assay and intra-assay relative standard deviation values were less than 10.8%, and the limits of quantitation for the proteo-specific peptides were quite low (1.0–5.0 μg/L). No appreciable matrix effects or carryover rates were observed. The extraction recoveries ranged from 93.8 to 128.7%, and the method was shown to be stable even when the samples were stored for 24 days. Prospective applications of this method in 909 participants also indicated good performance with a sensitivity of 100% and a specificity of 99.6%.
Conclusions
We have developed a fast, high throughput and reliable method for screening of β-thalassemia and hemoglobinopathy in children and adults, which is expected to be used as a first-line screening assay.
Funding source: Natural Science Foundation Project of Chongqing
Award Identifier / Grant number: cstc2021jcyj-msxmX0003
Funding source: Chongqing medical scientific research project (Joint project of Chongqing Health Commission and Science and Technology Bureau)
Award Identifier / Grant number: 2020FYYX189
Funding source: Clinical Research Project of Children’s Hospital of Chongqing Medical University
Award Identifier / Grant number: YBXM-2019-16
Acknowledgments
We thank all the participants for their cooperation and sample contributions. Present study is part of the postdoctoral program of Chongqing Medical University and Fuling Hospital affiliated to Chongqing University (Ziwei Li).
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Research funding: This study was supported by Clinical Research Project of Children’s Hospital of Chongqing Medical University (YBXM-2019-16) and the Natural Science Foundation Project of Chongqing (cstc2021jcyj-msxmX0003), partially supported by Chongqing medical scientific research project (Joint project of Chongqing Health Commission and Science and Technology Bureau, 2020FYYX189).
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Competing interests: Authors state no conflict of interest.
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Informed consent: Informed consent was obtained from all individuals included in this study.
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Ethical approval: The study was approved by the Institutional Ethical Review Boards of Children’s Hospital, Chongqing Medical University (No.2018-64).
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Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2022-0706).
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