Electrospray triple quadrupole mass spectrometry guides pathologists to suggest appropriate molecular testing in the identification of rare hemoglobin variants

Background: The presumptive diagnosis of hemoglobinopathies relies on routine tests such as Complete Blood Count (CBC), peripheral blood smear, Liquid Chromatography (LC), and Capillary Electrophoresis (CE), along with clinical findings. Pathologists suggest molecular sequencing of HBA and HBB genes to correlate blood picture with clinical findings in order to identify unknown rare haemoglobin (Hb) variants or variants that coelute with Hb. This paper presents a low-resolution mass spectrometry (MS)-based method for presumptive identification of variants that eluted in zone 12 of CE, followed by molecular sequencing of the HBB gene for a definitive diagnosis of hemoglobinopathies. Methods: Eight patient samples with a variant peak in zone 12 of CE (Sebia) were analyzed using MS. The mass-to-charge ratio (m/z) observed was deconvoluted to determine the mass of Hb variants. The β variants were subsequently confirmed through molecular sequencing. Results: Based on the intact mass of the variants, there were two samples of the α variant (α + 58 Da and α + 44 Da), and six samples of the β variant. Out of these six β variant samples, three were the β + 58 Da variant, and three were the β + 30 Da variant. By correlating the intact mass information with the CE pattern and considering the ethnicity of the patients, it was presumed that the α variants were HbJ Meerut (α + 58 Da, x-axis 102) and HbJ Paris-I (α + 44 Da, x-axis 80). Molecular analysis confirmed the identity of β variants as Hb Rambam/HbJ Cambridge, HbJ Bangkok (+58 Da), and Hb Hofu (+30 Da). Conclusion: The mass information of Hb variants obtained using Electrospray triple quadrupole MS assists pathologists in recommending the appropriate molecular sequencing for identifying unknown variants.