A novel method for quantification of human hemoglobin from dried blood spots by use of tandem mass spectrometry.
Quantification of human hemoglobin (Hb) is essential for diagnosis of anemia, especially for screening for thalassemia and sickle cell disease. The main methods currently used for quantification of Hb, including spectrophotometry, fluorimetry, and electrochemical assays, are all based on the structural integrity of Hb, which could be affected by hemolysis and degradation. When used for disease screening, whole blood specimens cannot meet requirements for sample collecting, transport, and storage. Here, we report a novel MS-MS method for quantification of Hb from dried blood spots (DBS) by use of a triple-quadrupole mass spectrometer. Proteospecific peptides from α-globin chains were selected after tryptic digestion. The precursor → product ion transitions of representative peptides were studied to identify the best choice with regard to sensitivity and chromatographic properties. For quantification, stable isotope-labeled peptides were used as internal standards. The concentration of Hb in each sample was obtained by calculation on the basis of established equations. The precision of the method was within 15 % and accuracy was in the range -7 to 13.0 %. Compared with routine clinical results obtained by use of the automated hematology analyzer (AHA) assay, the correlation, r (2), was >0.993. When used for determination of anemia levels the sensitivity of the assay was 95.7 % and specificity 96.5 %. Our new approach for quantification of the concentration of Hb from DBS is feasible, and precision is acceptable. The method could be used for determination of anemia levels when screening for hemoglobin disorders. Graphical Abstract Quantification of human hemoglobin from digested dried blood spot samples using tandem mass spectrometry.
Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, 136 2nd Zhongshan Rd, 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.
Chaowen
C
Yu
Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, 136 2nd Zhongshan Rd, 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.
Juan
J
Zhang
Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, 136 2nd Zhongshan Rd, 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.
Zhaojian
Z
Yuan
Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, 136 2nd Zhongshan Rd, 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.
Hao
H
Liu
Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, 136 2nd Zhongshan Rd, 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.
Xingbin
X
Wang
Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, 136 2nd Zhongshan Rd, 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.
Ming
M
Wang
Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, 136 2nd Zhongshan Rd, Chongqing, 400014, China. zoulin74@126.com.|Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing, 400014, China. zoulin74@126.com.|Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing, 400014, China. zoulin74@126.com.|Key Laboratory of Pediatrics in Chongqing, Chongqing, 400014, China. zoulin74@126.com.
Lin
L
Zou
D000328:Adult; D000595:Amino Acid Sequence; D002648:Child; D059788:Dried Blood Spot Testing / Q000379:methods*; D005260:Female; D006454:Hemoglobins / Q000032:analysis*; D006801:Humans; D007231:Infant, Newborn; D057230:Limit of Detection; D008297:Male; D008969:Molecular Sequence Data; D053719:Tandem Mass Spectrometry / Q000379:methods*
D006454:Hemoglobins
Dried blood spot (DBS); Hemoglobin; MS–MS; Mass spectrometry; Newborn screening