Newborn screening for sickle cell disease using tandem mass spectrometry.

BACKGROUND Neonatal screening programs for sickle cell disease are now widespread in North American and European countries. Most programs apply isoelectric focusing or HPLC to detect hemoglobin variants. Because tandem mass spectrometry (MS/MS) is being used for screening of inherited metabolic disorders and allows protein identification, it was worth testing for hemoglobinopathy screening. METHODS We minimized sample preparation and analysis times by avoiding prior purification, derivatization, or separation. We developed a tryptic digestion methodology to screen for the main clinically important variants (Hb S, Hb C, and Hb E) and beta-thalassemia. To ensure proper discrimination between homozygote and heterozygote variants, we selected 4 transitions with good signal intensities for each specific peptide and calculated variant/Hb A ratios for each. Method validation included intra- and interseries variability, carryover, and limit of detection. We also performed a comparative study with isoelectric focusing results on 2082 specimens. RESULTS Intraassay imprecision values (CVs) varied between 2.5% and 30.7%. Interassay CVs were between 6.3% and 23.6%. Carryover was <0.03%, and the limit of detection was fixed at 1% of Hb S. According to the MS/MS settings (detection of Hb S, Hb C, Hb E, and beta-globin production defects), the comparative study did not yield any discrepant results between the 2 techniques. CONCLUSIONS MS/MS is a reliable method for hemoglobinopathy neonatal screening.