A Mass Spectrometry-Based Multiplexed Targeted Assay for Detection of Hemoglobinopathies from Dried Blood Spots

Hemoglobinopathies are the most common inherited disorders worldwide. Accurate analysis of hemoglobin variants is critical for diagnosis of hemoglobinopathies. Although high-performance liquid chromatography and capillary zone electrophoresis are widely used as screening tools, they possess inherent ambiguities that often preclude accurate detection of hemoglobin variants. The goal was to develop and optimize a sensitive and specific mass spectrometry-based assay for screening and diagnosis of hemoglobinopathies. A catalog of canonical globin-chain specific peptides as well as mutant peptides corresponding to common hemoglobin variants was generated, and their corresponding heavy synthetic peptide versions were used as internal standards for quantification and calculation of globin chain ratios. Targeted mass spectrometry analysis was performed by coupling liquid chromatography to a triple quadrupole mass spectrometer, which is the most common mass spectrometer used in clinical diagnostics. Dried blood spots from a cohort of 716 individuals (including 211 patients with hemoglobinopathy) were analyzed. The α:β-globin ratios showed a significant difference between normal patients and patients with β-thalassemia, particularly when the disease was homozygous or admixed with structural variants (compound heterozygous). The method presented here permits identification of variants in their homozygous, heterozygous, or compound heterozygous states. The intra-assay and interassay precision CV were both <20%. We envision that such mass spectrometry-based assays could be used as first-line screening assay for hemoglobin variants, including sickle cell disease as well as thalassemias.