The development of mass spectrometry-based approaches for the diagnosis of hemoglobinopathies

• Main Author: Radi, Krisztina
• Published: University of Warwick 2013
• Subjects: 570; RC Internal medicine
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.589819

Hemoglobinopathies are disorders of the protein hemoglobin (Hb), one of the most common inherited disorders, and pose an increasing healthcare problem. Worldwide, approximately 200 million people have hemoglobinopathies (Hartwell et al. 2005), and whilst the majority of them are clinically silent, some are life-threatening. Screening programs for hemoglobinopathies traditionally employ chromatography and electrophoretic techniques, which only provide presumptive identification of clinically important hemoglobin disorders. The definitive characterisation of these disorders requires protein sequence elucidation or DNA analysis. The development of a rapid population screening method for hemoglobinopathies, which could also provide definitive diagnosis of a disorder, is of significant interest to the healthcare profession. Mass spectrometry (MS) is currently used to routinely characterise variants but not as a validated clinical diagnostic tool. The work undertaken addresses the increasing demand for population screening. Different mass spectrometry-based approaches to hemoglobinopathy detection have been developed based on previously described procedures found in literature. The methods, for intact globin chain analysis and targeted variant specific peptide analysis, have been optimised and evaluated using clinical blood samples on a triple quadrupole system. Intact globin chain analysis and targeted tryptic peptide screening methods have been evaluated in a clinical trial analysing 2017 blood samples to identify clinically significant variants routinely screened as a requirement by the NHS Screening programme and to quantify minor globin chains (HbA2 and HbF) suitable for the diagnosis of thalassemic and other disorders. This work was carried out in a three month mirrored testing experiment against existing clinical laboratory methods. The results obtained showed an excellent correlation to values from current clinical methods, as all clinically significant variants were detectable by MS methods. In addition, MS was able to detect Hb variants undetected by electrophoretic or chromatographic methods and highlighted some issues with the current phenotypic methods which can lead to misdiagnosis. A top-down ETD method utilising an iontrap mass spectrometer has been developed and applied to provide fragmentation for positive identification of known hemoglobin variants. This method was evaluated by analysing 1000 samples in a pilot study as a sideproject to the clinical trial. The generation of an automated report with the detection of clinically significant variants and minimal sample preparation are some of the benefits associated with this approach. This method has been shown to meet the majority of requirements for a rapid diagnostic screening method and has the potential to become a validated tool in the clinical laboratory screening in the near future.