Tandem mass spectrometric determination of purine metabolites and adenosine deaminase activity for newborn screening of ADA–SCID
Abstract
Background: Screening newborns for severe combined immunodeficiency (SCID) aims for early identification and treatment of the affected newborns. Adenosine deaminase (ADA) deficiency, a defect in the purine metabolic pathway, is a major cause of SCID and is characterized by the accumulation of adenosine (Ado) and deoxyadenosine (dAdo) in dried blood spots (DBSs). If left untreated, infants with this disorder are at risk of life-threatening infections. Analysis of T-cell receptor excision circles (TRECs) in DBS samples is the gold-standard screening method. However, TREC analysis is insufficient to determine SCID etiology, and a fraction of ADA–SCID may not be detected.
Methods: We used the original DBS screening sample to measure Ado, dAdo, and ADA activity. Erythro-9-(2-hydroxy-3-nonyl) adenine was used as an ADA inhibitor to imitate ADA deficiency, making it possible to create quality control material with pathological enzyme activity and metabolite levels. Quantification was achieved by tandem mass spectrometric analysis with a run time of 2.5 min.
Results: The 95th percentile reference intervals (n = 588) of Ado and dAdo were 0.9–3.0 and 0.1–0.4 µmol/L, respectively. The 95th percentile reference interval (n = 200) of ADA activity using 13C10, 15N5 Ado and 15N5 dAdo as substrates were 0.8–1.6 and 0.4–0.7 pmol/DBS, respectively. In confirmed ADA patients (n = 4), Ado and dAdo were significantly elevated, whereas ADA activity was almost absent.
Conclusion: These novel methods are applied, in our lab, to samples with low TRECs, with no false negative or false positives encountered to date. The potential of using these methods as a primary screening approach for ADA–SCID is in the process of validation.
Statement of novelty: New mass spectrometric methods to simultaneously measure adenosine, deoxyadenosine, guanosine, and deoxguanosine, as well as ADA activity in neonatal DBS samples have been developed. This methodology highlights the metabolic nature of ADA–SCID and complements TREC analysis by providing additional biochemical information.
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LymphoSign Journal
Volume 2 • Number 3 • September 2015
Pages: 135 - 145
History
Received: 29 December 2014
Accepted: 18 February 2015
Accepted manuscript online: 10 March 2015
Version of record online: 10 March 2015
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Osama Y. Al-Dirbashi, Svetlana Ogrel, Nathan McIntosh, Lauren Higgins, Christine McRoberts, Larry Fisher, Dennis E. Bulman, Michael T. Geraghty, and Pranesh Chakraborty. 2015. Tandem mass spectrometric determination of purine metabolites and adenosine deaminase activity for newborn screening of ADA–SCID. LymphoSign Journal.
2(3): 135-145. https://doi.org/10.14785/lpsn-2014-0024
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