title: A novel tandem mass spectrometry method for first-line screening of mainly beta-thalassemia from dried blood spots.
abstract: Traditional methods for thalassemia screening are time-consuming and easily affected by cell hemolysis or hemoglobin degradation in stored blood samples. Tandem mass spectrometry (MS/MS) proved to be an effective technology for sickle cell disorders (SCD) screening. Here, we developed a novel MS/MS method for β-thalassemia screening from dried blood spots (DBS). Stable isotopic-labeled peptides were used as internal standards for quantification and calculation of the α:β-globin ratios. We used the α:β-globin ratio cutoffs to differentiate between normal individuals and patients with thalassemia. About 781 patients and 300 normal individuals were analyzed. The α:β-globin ratios showed significant difference between normal and β-thalassemia patients (P<0.01), particularly when the disease was homozygous or double heterozygous with another α- or β-thalassemia mutation. In the parallel study, all cases screened for suspected thalassemia from six hundred DBS samples by using this MS/MS method were successfully confirmed by genotyping. The intra-assay and inter-assay CVs of the ratios ranged from 2.4% to 3.9% and 4.7% to 7.1%, and there was no significant sample carryover or matrix effect for this MS/MS method. Combined with SCD screening, this MS/MS method could be used as a first-line screening assay for both structural and expression abnormalities of human hemoglobin.
  
  
  
  Traditional methods for thalassemia screening were depending on the structural integrity of tetramers and could be affected by hemolysis and degradation of whole blood samples, especially when stored. We used proteospecific peptides produced by the tryptic digestion of each globin to evaluate the production ratio between α- and β-globin chains, which turned out to be quite stable even when stored for more than two months. Though most of the peptides were specific to α-globin or β-globin, we only chose four most informative peptides and its stable isotopic-labeled peptides as internal standards for analysis, which could obtain a high accuracy. Currently, we are the first to address the application of MS/MS for thalassemia screening, when combined with SCD screening, this MS/MS method could be used as a first-line screening assay for both structural and expression abnormalities of human hemoglobin.
authors.affiliation: Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, Chongqing 400014, China; Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing 400014, China; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing 400014, China; Key Laboratory of Pediatrics in Chongqing, Chongqing 400014, China.
authors.forename: Chaowen
authors.initials: C
authors.lastname: Yu
authors.affiliation: Center for Neonatal Screening, Meizhou Maternal and Child Health Care Hospital, Guangdong 514000, China.
authors.forename: Shuodan
authors.initials: S
authors.lastname: Huang
authors.affiliation: Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, Chongqing 400014, China; Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing 400014, China; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing 400014, China; Key Laboratory of Pediatrics in Chongqing, Chongqing 400014, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Wang
authors.affiliation: Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, Chongqing 400014, China; Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing 400014, China; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing 400014, China; Key Laboratory of Pediatrics in Chongqing, Chongqing 400014, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, Chongqing 400014, China; Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing 400014, China; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing 400014, China; Key Laboratory of Pediatrics in Chongqing, Chongqing 400014, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Liu
authors.affiliation: Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, Chongqing 400014, China; Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing 400014, China; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing 400014, China; Key Laboratory of Pediatrics in Chongqing, Chongqing 400014, China.
authors.forename: Zhaojian
authors.initials: Z
authors.lastname: Yuan
authors.affiliation: Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, Chongqing 400014, China; Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing 400014, China; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing 400014, China; Key Laboratory of Pediatrics in Chongqing, Chongqing 400014, China.
authors.forename: Xingbin
authors.initials: X
authors.lastname: Wang
authors.affiliation: Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, Chongqing 400014, China; Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing 400014, China; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing 400014, China; Key Laboratory of Pediatrics in Chongqing, Chongqing 400014, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: He
authors.affiliation: Center for Neonatal Screening, Hainan Maternal and Child Health Care Hospital, Haikou 570206, China. Electronic address: wj59726@sina.com.
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.affiliation: Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, Chongqing 400014, China; Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing 400014, China; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing 400014, China; Key Laboratory of Pediatrics in Chongqing, Chongqing 400014, China. Electronic address: zoulin74@126.com.
authors.forename: Lin
authors.initials: L
authors.lastname: Zou
mesh_terms: D001800:Blood Specimen Collection / Q000379:methods; D016022:Case-Control Studies; D005838:Genotype; D006801:Humans; D008403:Mass Screening / Q000379:methods; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D055542:alpha-Globins / Q000032:analysis; D055544:beta-Globins / Q000032:analysis; D017086:beta-Thalassemia / Q000097:blood / Q000175:diagnosis*
chemical_list: D055542:alpha-Globins; D055544:beta-Globins
keywords: Beta-thalassemia; Disease screening; Dried blood spots (DBS); Hemoglobin; Tandem mass spectrometry (MS/MS)