A reliable and high throughput HPLC-HRMS method for the rapid screening of β-thalassemia and hemoglobinopathy in dried blood spots.

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 (β/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.