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Received on March 16, 2007
Accepted on May 15, 2007
Hematology |
1 Blood Sciences Delivery Unit, St. Thomas' Hospital, Guy's and St. Thomas' NHS Foundation Trust, London, United Kingdom
2 WellChild Laboratory, Evelina Children's Hospital, Guy's and St. Thomas' NHS Foundation Trust, London, United Kingdom
3 WellChild Laboratory, King's College London, Evelina Children's Hospital, London, United Kingdom
* To whom correspondence should be addressed. E-mail: neil.dalton{at}kcl.ac.uk.
Background: Peptide-based analysis of whole blood using electrospray tandem mass spectrometry (MSMS) in multiple reaction monitoring (MRM) mode enables rapid detection and sequence confirmation of clinically significant hemoglobin (Hb) variants. We applied a similar, quantitative approach to the measurement of 
:
-globin peptide ratios as potential surrogate markers of HbA2, a biomarker used in population screening for -thalassemia trait.
Methods: We studied 163 blood samples with normal HbA2 (%), 105 with increased HbA2, 43 with -chain variants, and 8 with Hb Lepore. All were tested by HPLC. The samples were also incubated with trypsin for 30 min at 37 °C for MSMS with flow injection analysis. MRMs for the - (T2, T3, and T14) and - (T2, T3, and T13) globin tryptic peptides were acquired for 1 min, and : peptide ratios were calculated. We used HPLC and MSMS to analyze 26 paired whole blood and dried blood spot samples after storage for 1, 8, and 29 days.
Results: Within- and between-assay imprecision values (CVs) were <6.1% and <8.4%, respectively, for the : peptide ratios. Digests were stable at 10 °C for 6 days. Significant correlations (P <0.0001) between MSMS :-globin peptide ratios and HPLC HbA2 allowed differentiation between increased HbA2 concentrations and concentrations within the reference interval and identification of Hb Lepore. This differentiation was repeatable by MSMS, but not by HPLC, after blood spot samples had been stored for 1 month.
Conclusion: This study validates the quantitative :-globin peptide ratio as a surrogate marker of HbA2 and demonstrates the potential of rapid peptide-based MSMS for multiplexed, high-throughput protein biomarker characterization and quantification.
The following articles in journals at HighWire Press have cited this article:
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  D. D. Mais, R. D. Gulbranson, and D. F. Keren The Range of Hemoglobin A2 in Hemoglobin E Heterozygotes as Determined by Capillary Electrophoresis Am J Clin Pathol, July 1, 2009; 132(1): 34 - 38. [Abstract] [Full Text] [PDF]
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 C. Turner, Y. Daniel, and R. N. Dalton Newborn Screening for Sickle Cell Disease through Use of Tandem Mass Spectrometry Clin. Chem., June 1, 2009; 55(6): 1243 - 1244. [Full Text] [PDF]
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T. N. Higgins, A. Khajuria, and M. Mack Quantification of HbA2 in Patients With and Without {beta}-Thalassemia and in the Presence of HbS, HbC, HbE, and HbD Punjab Hemoglobin Variants: Comparison of Two Systems Am J Clin Pathol, March 1, 2009; 131(3): 357 - 362. [Abstract] [Full Text] [PDF]
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 A Streetly, R Latinovic, K Hall, and J Henthorn Implementation of universal newborn bloodspot screening for sickle cell disease and other clinically significant haemoglobinopathies in England: screening results for 2005-7 J. Clin. Pathol., January 1, 2009; 62(1): 26 - 30. [Abstract] [Full Text] [PDF]
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F. Boemer, O. Ketelslegers, J.-M. Minon, V. Bours, and R. Schoos Newborn Screening for Sickle Cell Disease Using Tandem Mass Spectrometry Clin. Chem., December 1, 2008; 54(12): 2036 - 2041. [Abstract] [Full Text] [PDF]
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S. O. Brennan Fifty-Eight Years of Hemoglobin Analysis Clin. Chem., January 1, 2008; 54(1): 8 - 10. [Full Text] [PDF]
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