title: 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.
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.
authors.forename: Giancarlo
authors.initials: G
authors.lastname: la Marca
authors.forename: Elisa
authors.initials: E
authors.lastname: Giocaliere
authors.forename: Sabrina
authors.initials: S
authors.lastname: Malvagia
authors.forename: Fabio
authors.initials: F
authors.lastname: Villanelli
authors.forename: Silvia
authors.initials: S
authors.lastname: Funghini
authors.forename: Daniela
authors.initials: D
authors.lastname: Ombrone
authors.forename: Maria
authors.initials: M
authors.lastname: Della Bona
authors.forename: Giulia
authors.initials: G
authors.lastname: Forni
authors.forename: Clementina
authors.initials: C
authors.lastname: Canessa
authors.forename: Silvia
authors.initials: S
authors.lastname: Ricci
authors.forename: Francesca
authors.initials: F
authors.lastname: Romano
authors.forename: Renzo
authors.initials: R
authors.lastname: Guerrini
authors.forename: Massimo
authors.initials: M
authors.lastname: Resti
authors.forename: Chiara
authors.initials: C
authors.lastname: Azzari
mesh_terms: D000328:Adult; D002853:Chromatography, Liquid; D059788:Dried Blood Spot Testing; D006801:Humans; D007231:Infant, Newborn; D015997:Neonatal Screening; D000081207:Primary Immunodeficiency Diseases; D011683:Purine-Nucleoside Phosphorylase / Q000097:blood / Q000172:deficiency* / Q000378:metabolism; D011686:Purine-Pyrimidine Metabolism, Inborn Errors / Q000097:blood* / Q000175:diagnosis / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D011683:Purine-Nucleoside Phosphorylase