The inclusion of ADA-SCID in expanded newborn screening by tandem mass spectrometry

Abstract

a b s t r a c tSevere combined immunodeficiency due to adenosine-deaminase defect (ADA-SCID) is usually deadly in childhood because of severe recurrent infections. When clinical diagnosis is done, permanent damages due to infections or metabolite accumulation are often present. Gene therapy, bone marrow transplantation or enzyme replacement therapy may be effective if started early. The aim of this study was to set-up a robust method suitable for screening with a minimized preparation process and with inexpensive running costs, for diagnosing ADA-SCID by tandem mass spectrometry. ADA-SCID satisfies all the criteria for inclusion in a newborn screening program.We describe a protocol revised to incorporate adenosine and 2-deoxyadenosine testing into an expanded newborn screening program. We assessed the effectiveness of this approach testing dried blood spots from 4 genetically confirmed early-onset and 5 delayed-onset ADA-SCID patients. Reference values were established on 50,000 healthy newborns (deoxyadenosine <0.09 mol/L, adenosine <1.61 mol/L). We also developed a second tier test to distinguish true positives from false positives and improve the positive predictive value of an initial abnormal result.In the first 18 months, the pilot project has identified a newborn with a genetically confirmed defect in adenosine deaminase (ADA) gene.The results show that the method having great simplicity, low cost and low process preparations can be fully applicable to a mass screening program.

Tandem mass spectrometric determination of purine metabolites and adenosine deaminase activity for newborn screening of ADA–SCID

“…In the early experiments, unlike Guo and dGuo, we noticed the immediate loss of Ado and dAdo upon adding standard purines to whole blood. A similar observation was also described by la Marca et al (2014a) who ascribed this to residual ADA activity and opted to use aqueous calibrators. In MS-MS based methods, the use of matrix-matched control materials is an acceptable approach to reduce the matrix effect on target analytes (Kilcoyne and Fux 2010).…”

“…ADA-SCID patients can be identified by measuring purine metabolites, namely Ado and dAdo, in DBS specimens. In the literature, analysis of these metabolites using MS-MS has been described; however, the published method does not allow for preparing control samples in whole blood, owing to residual enzyme activity (Azzari et al 2011;la Marca et al 2013;la Marca et al 2014a). Further, the use of a single 13 C labelled Ado IS in the published method is inappropriate, as it shares the same mass transition with the natural Ado isotope (la Marca et al 2014a).…”

“…In the literature, analysis of these metabolites using MS-MS has been described; however, the published method does not allow for preparing control samples in whole blood, owing to residual enzyme activity (Azzari et al 2011;la Marca et al 2013;la Marca et al 2014a). Further, the use of a single 13 C labelled Ado IS in the published method is inappropriate, as it shares the same mass transition with the natural Ado isotope (la Marca et al 2014a). Therefore, we sought to develop a method that employs spiked blood controls and extend it to encompass other purines such as Guo and dGuo, the markers for purine nucleoside phosphorylase deficiency within the same run (Chantin et al 1996;la Marca et al 2014b).…”

“…Owing to the inability of the assay for TRECs to provide information about Ado and dAdo, which are present at elevated levels in patients with ADA deficiency, another method of analysis for these compounds in DBS specimens is warranted. These markers were detected in DBS by tandem mass spectrometry (MS-MS) and were shown to considerably improve newborn screening for ADA-SCID by introducing an etiologic focus (Azzari et al 2011;la Marca et al 2014a). …”

Mass spectrometry in clinical chemistry: the case of newborn screening

“…People working in newborn screening field understood the possibility to pass from one DBS for one test and one disorder to one DBS for one multiplex test for many disorders. In fact, today MS/MS can easily identify and quantify in a 2 or less minutes' run -several metabolites such as acylcarnitines, aminoacids, succinylacetone [3,4] and more recently some purines [5][6][7][8]. Nowadays, including pilot projects regionally ruled and structured national NBS programs, many labs all around the world screen for more than 30 or more IEM with a single test.…”