Mass Spectrometry-based Study Of Screening For β-thalassemia And Hemoglobinopathy

Objective:The traditional methods forβ-thalassemia screening usually rely on the structural integrity of hemoglobin,which can be affected by the hemolysis of red blood cells and Hb degradation.The conventional laboratory tests with the defects of low sensitivity and specificity,poor repeatability,high costs and tedious operation are not suitable for disease screening.This study is intended to find a novel method,which is simple,maneuverable,low cost but high-throughput for detectingβ-thalassemia,and to explore the clinical application of dried blood filter card samples for the disease using a high pressure liquid chromatography combined high-resolution mass spectrometry approach.The aim of this project is to expand the conditions checked by prenatal screening and newborn screening programs in China,and to achieve the ultimate goal of reducing birth defects of globin synthesis aplastic anemia and improve the overall quality of population.Methods:We used HPLC-HRMS to establish a method for micro hemoglobin extraction from dried blood spot（DBS）samples.With the use of tryptic digestion,the marker peptides of mainlyαglobin、βglobin、γglobin andδglobin were produced from DBS.We optimized the gradient parameters of the Ultimate 3000 chromatographic system.C18 UHPLC column was used to separate,purify and concentrate the peptides generated form DBS samples.HRMS were used to screen the proteo-specific peptides.The target-MS²acquiring model was chosen to analyze the peptide fragments and the appropriate mass transitions were selected.The relative concentrations of the proteo-specific peptides were determined by using stable isotope-labeled peptides as internal standards（IS）.To obtain a better mass spectrum peak,we optimized HRMS ion source parameters and Trace Finder quantitative software parameters.Based on HPLC-HRMS method to detection samples,the ratio of the peak area of the selected peptide to IS was determined to calculate theδ/βandβM/βratio,which can reflect the ratio of Hb A₂/Hb A in the samples.To generate the reference interval ofβ-thalassemia and hemoglobinopathy,the globin chain ratios were calculated in accordance with equations F1 and F2.Receiver operating characteristic curve（ROC）was performed to determine the appropriate cutoffs for different types of thalassemia and hemoglobinopathies.To assess the HPLC-HRMS method,we investigated the precision,limits of quantification（LOQ）,matrix effect,carried contamination,sample stability,interference experiment,standard curve,the recovery of the peptides.To validate the screening cutoffs and the performance of HPLC-HRMS method,the sensitivity and specificity of the HPLC-HRMS method were finally evaluated by comparing with NGS.Results:Totally 699 confirmedβ-thalassemia patients,25hemoglobinopathy patients and 629 normal control individuals were recruited for method construction.A method for extracting micro hemoglobin from dry blood spot（DBS）samples was successfully established by optimizing pre-treatment methods.Specific peptides ofβ-thalassemia and hemoglobinopathy were screened and identified.The relative concentrations of the proteo-specific peptides were determined by using HPLC-HRMS method,and then a HPLC-HRMS based method was developed to qualitatively and quantitatively analyze the specific peptides,and use forβthalassemia and hemoglobinopathy screening.Method evaluation has confirmed the performance of the new HPLC-HRMS method.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（1.0～5.0μg/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.The ROC analysis showed that the appropriate globin ratio cutoffs with the highest sensitivity and specificity were determined:β⁺thalassemia,δ/β≥0.075,the sensitivity and specificity reached 99.27%and 99.36%;β⁰thalassemia,δ/β≥0.109,the sensitivity and specificity reached 100%;and hemoglobinopathy,βM/β≥0.000,the sensitivity and specificity reached 100%.NGS was used as a“golden stander”method to assess the performance of the HPLC-HRMS method.The clinical application of the HPLC-HRMS method was determined to have a sensitivity of 100%and a specificity of 99.6%in the present recruited group.Conclusions:We have developed a fast,high throughput and reliable method for screening ofβ-thalassemia and hemoglobinopathy in children and adults,which is expected to be used as a first-line screening assay in the future.