Direct cord blood LAMP colorimetric phenol red assay for detecting α⁰-thalassemia (SEA deletion); the validation and post-natal screening in Thailand

Abstract

Post-natal or newborn screening for thalassemia and hemoglobinopathies is useful for genetic counseling and managing thalassemia in children. We characterized thalassemia genotypes in newborns from the eastern part of Thailand. The results demonstrated a high heterogeneity of thalassemia and hemoglobinopathies with seventeen genotypes. We focused on α⁰- thalassemia (Southeast Asian [SEA] deletion) in this study. We developed and validated the loop-mediated isothermal amplification (LAMP) colorimetric assay for detecting α⁰- thalassemia (SEA deletion) using simple direct cord blood sampling compared to genomic DNA. A total of 160 cord blood samples were evaluated with the LAMP assay. The sensitivity and specificity of the LAMP colorimetric assay for α⁰-thalassemia (SEA deletion) using direct cord blood showed 100% (6/6 x 100) and 98.05% (151/154 x 100) whereas, genomic DNA showed 100% (6/6 x 100) and 100% (154/154 x 100), respectively. Moreover, we demonstrated other simple screening tools for α⁰-thalassemia with %Hb Bart’s, MCV, and MCH values and found that these parameters were not diagnostic in our samples. The direct cord blood with colorimetric LAMP assay is simple, rapid, and does not require a post-LAMP step compared to conventional PCR. These techniques could be applied in post-natal or large population screening for α⁰-thalassemia (SEA deletion). Finally, this could support early prevention of complications, early management, genetic counseling for α-thalassemia disease in children, or a long-term prevention and control program of severe thalassemia in Thailand.

Keywords:

• Hemoglobinopathy
• α-thalassemia
• loop-mediated isothermal amplification colorimetric assay
• screening
• prevention
• biomedical

Acknowledgements

The authors thank the medical staff, residents and nursing at Obstetrics and Gynecology, HRH Princess Mahachakri Sirindhorn Medical Center for cord blood collection. The Zybio Nucleic Acid extraction kit was supported by Z-Medic Co.,LTD.

Ethical approval

Ethical approval for this study was obtained from the Institutional Review Board of Srinakharinwirot University, Thailand (SWUEC/E/M-023/2564).

Author contributions

WJ was responsible for acquisition of the grant, the design of the study, performed the research, analyzed data, and drafted the manuscript. RK involved with design of the study, performed the experiment, and drafted the manuscript. AN and WL was responsible for cord blood specimen collection and the design of the study. PP involved with routine laboratory investigation. All authors approved the final version.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Additional information

Funding

This study was supported by a research grant from HRH Princess Mahachakri Sirindhorn Medical Center, Faculty of Medicine, Srinakharinwirot University (Contract No 081/2565).