Development and validation of a 2nd tier test for identification of purine nucleoside phosphorylase deficiency patients during expanded newborn screening by liquid chromatography-tandem mass spectrometry

Giancarlo la Marca; Elisa Giocaliere; Sabrina Malvagia; Fabio Villanelli; Silvia Funghini; Daniela Ombrone; Maria Della Bona; Giulia Forni; Clementina Canessa; Silvia Ricci; Francesca Romano; Renzo Guerrini; Massimo Resti; Chiara Azzari

doi:10.1515/cclm-2015-0436

Artikel

Development and validation of a 2nd tier test for identification of purine nucleoside phosphorylase deficiency patients during expanded newborn screening by liquid chromatography-tandem mass spectrometry

Giancarlo la Marca , Elisa Giocaliere , Sabrina Malvagia , Fabio Villanelli , Silvia Funghini , Daniela Ombrone , Maria Della Bona , Giulia Forni , Clementina Canessa , Silvia Ricci , Francesca Romano , Renzo Guerrini , Massimo Resti und Chiara Azzari

Veröffentlicht/Copyright: 14. Oktober 2015

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Aus der Zeitschrift Clinical Chemistry and Laboratory Medicine (CCLM) Band 54 Heft 4

Abstract

Background: Purine nucleoside phosphorylase (PNP) deficiency has been recently introduced in the newborn screening program in Tuscany. In order to improve the PNP screening efficiency, we developed a 2nd tier test to quantify PNP primary markers deoxyguanosine (dGuo) and deoxyinosine (dIno).

Methods: Dried blood spots (DBS) samples were extracted with 200 μL of methanol and 100 μL of water (by two steps). Internal standards were added at a final concentration of 10 μmol/L. After extraction, samples were analysed by LC-MS/MS. The chromatographic run was performed in gradient mode by using a Synergi Fusion column.

Results: The assay was linear over a concentration range of 0.05–50 μmol/L (R²>0.999) for dGuo and 0.5–50 μmol/L (R²>0.998) for dIno. Intra- and interassay imprecision (mean CVs) for dIno and dGuo ranged from 2.9% to 12%. Limit of quantitaion (LOQ) were found to be 0.05 μmol/L and 0.5 μmol/L for dGuo and dIno, respectively. The reference ranges, obtained by measuring dGuo and dIno concentrations on DBS, were close to zero for both biomarkers. Moreover, DBS samples from seven patients with confirmed PNP were retrospectively evaluated and correctly identified.

Conclusions: The LC-MS/MS method can reliably measure dIno and dGuo in DBS for the diagnosis of PNP. Validation data confirm the present method is characterised by good reproducibility, accuracy and imprecision for the quantitation of dIno and dGuo. The assay also appears suitable for use in monitoring treatment of PNP patients.

Keywords: 2nd tier test; dried blood spot; newborn screening; purine nucleoside phosporilase; purine nucleoside phosphorylase deficiency; severe combined immunodeficiency; tandem mass spectrometry

Corresponding author: Giancarlo la Marca, Newborn Screening, Biochemistry and Pharmacology Laboratories, Clinic of Pediatric Neurology, Meyer University Children’s Hospital, Viale Pieraccini 24, 50139 Florence, Italy, Phone: +390555662988; Fax: +390555662849, E-mail: g.lamarca@meyer.it; giancarlo.lamarca@unifi.it; and Department of Neurosciences, Psychology, Pharmacology and Child Health, University of Florence, Italy

Acknowledgments

The authors want to thank Prof. Micheal Hershfield from the department of Medicine, Duke University, Durham, USA; Dr. Carsten Speckmann from Center for Chronic Immunodeficiences, University of Freiburg, Germany; Dr. Andrew R. Gennery from the Insitute of Cellular Medicine, Newcastle University, UK; Dr. Fatih Celmeli from Antalya Education and Research Hospital, Antalya, Turkey for providing PNP patient’ samples.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: None declared.

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Received: 2015-5-8

Accepted: 2015-8-28

Published Online: 2015-10-14

Published in Print: 2016-4-1

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https://doi.org/10.1515/cclm-2015-0436

Schlagwörter für diesen Artikel

2nd tier test; dried blood spot; newborn screening; purine nucleoside phosporilase; purine nucleoside phosphorylase deficiency; severe combined immunodeficiency; tandem mass spectrometry