Screening blood spots for inborn errors of metabolism by electrospray tandem mass spectrometry with a microplate batch process and a computer algorithm for automated flagging of abnormal profiles Mohamed S. Rashed, 1 * Martin P. Bucknall, 1 Douglas Little, 1 Amin Awad, 1 Minnie Jacob, 1 Mohamed Alamoudi, 1 Mona Alwattar, 1 and Pinar T. Ozand 1,2 Metabolic profiling of amino acids and acylcarnitines from blood spots by automated electrospray tandem mass spectrometry (ESI-MS/MS) is a powerful diagnos- tic tool for inborn errors of metabolism. New ap- proaches to sample preparation and data interpretation have helped establish the methodology as a robust, high-throughput neonatal screening method. We intro- duce an efficient 96-well-microplate batch process for blood-spot sample preparation, with which we can obtain high-quality profiles from 500-1000 samples per day per instrument. A computer-assisted metabolic pro- filing algorithm automatically flags abnormal profiles. We selected diagnostic parameters for the algorithm by comparing profiles from patients with known metabolic disorders and those from normal newborns. Reference range and cutoff values for the diagnostic parameters were established by measuring either metabolite con- centrations or peak ratios of certain metabolite pairs. Rigorous testing of the algorithm demonstrates its out- standing clinical sensitivity in flagging abnormal pro- files and its high cumulative specificity. INDEXING TERMS: amino acids • organic acidemia • defects of fatty acid oxidation • inherited disorders • neonatal screening Inborn errors of metabolism (IEMs) 3 and other inherited Mendelian disorders are common in Saudi Arabia and throughout the Middle East, presumably because of the relatively high rates of consanguinity [1– 4]. Even in segregated communities, IEMs are estimated to account for as much as 20% of disease among full-term neonates not known to have been at risk and may affect as many as 1 in 5000 live births [5, 6]. Many of the IEMs carry serious clinical consequences to the affected neonate or young infant, including mild or severe mental retardation, phys- ical handicap, and even fatality. Although early diagnosis for some of these disorders has proven very effective in treatment or management, neonates are screened for only a handful of diseases, even in the developed world [7–11]. For 30 years in developed countries, the Guthrie qualitative bacterial inhibition test for phenylalanine in blood spots has formed the basis for successful national neonatal screening programs for phenylketonuria (PKU) [12]. Similar methods have been adopted on a limited scale for diseases such as maple syrup urine disease, isolated hypermethioninemia, homocystinuria, and galac- tosemia [10, 13]. The more comprehensive quantitative determination of plasma amino acids by HPLC is usually reserved for selective screening of high-risk infants, for confirmation of neonatal screening results, for follow-up of treatment, and for identification of other aminoacid- opathies [14, 15]. Departments of 1 Biological and Medical Research and 2 Pediatrics, King Faisal Specialist Hospital and Research Centre, PO Box 3354, Riyadh 11211, Saudi Arabia. *Author for correspondence. Fax 966-1-442-7858; e-mail rashed@kfshrc. edu.sa. Received July 29, 1996; revised and accepted January 8, 1997. 3 Nonstandard abbreviations: IEMs, inborn errors of metabolism; PKU, phenylketonuria; CAMPA, computer-assisted metabolic profiling algorithm; ESI-MS/MS, electrospray tandem mass spectrometry; FAB-MS/MS, fast atom bombardment tandem mass spectrometry; FDI, functions of diagnostic inter- est; MDI, masses of diagnostic interest; RDI, (peak) ratios of diagnostic interest; and GA-I/II, glutaric acidemia type I/II. Clinical Chemistry 43:7 1129 –1141 (1997) Molecular Pathology 1129 Downloaded from https://academic.oup.com/clinchem/article-abstract/43/7/1129/5640906 by guest on 24 July 2020