Progress in expanded newborn screening for metabolic conditions by LC‐MS/MS in Tuscany: Update on methods to reduce false tests

Abstract

We report on our 6-year experience of expanded newborn screening by tandem mass spectrometry in Tuscany (Italy), the first Italian Region to screen all newborns for more than 40 inborn errors of metabolism: organization, diseases observed and updates on methods to reduce false-positive and false-negative tests are described. Blood collection is recommended between 48 and 72 h of life. Blood spots are sent daily by courier to laboratory. When a positive result occurs, two subsequent procedures are followed: for disorders with possible acute metabolic decompensation, the baby is immediately recalled and clinical examinations and confirmatory tests are performed; for the other disorders, the nursery provides for a second blood spot. If the test is positive, clinical examinations and confirmatory tests are performed. In both cases, if confirmatory tests are positive, a treatment and a follow-up programme are started. Up to now, spots from 160 000 infants have been analysed and 80 affected patients have been identified (disorders of amino acids, organic acids and fatty acids metabolism). We describe adjustments to cut-off values, the introduction of a second-tier test for propionic acidaemia and for methylmalonic aciduria, the inclusion of succinylacetone in the panel of metabolites, and protocols for premature infants and for newborns on parenteral nutrition or transfused. These changes resulted in a reduction in recalls from 1.37% to 0.32% and consequently of working time and parental stress. Avoiding false-negatives by using more specific markers and minimizing the false-positive rate with second-tier testing is important for a successful newborn screening programme.

Diagnosis of immunodeficiency caused by a purine nucleoside phosphorylase defect by using tandem mass spectrometry on dried blood spots

“…Sample preparation for routine screening tests is exactly as reported by la Marca et al, 13 with the exception of the butylation procedure, which is not carried out.…”

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

“…Mass spectrometry is extensively used in newborn screening programs for analysis of metabolites from dried blood spots (DBS) collected a few days after birth, but among the detected metabolites those due to ADA deficiency have not been included so far [19,20].…”

“…In the first 6 months of the pilot project, DBS were processed in parallel by both the methods reported by la Marca et al [20] and the new screening method reported by Azzari et al [21] which is performed without butylation procedure and with the addiction of Ado and dAdo IS, both at 1 mol/L for each well.…”

Dried blood spot sampling in combination with LC‐MS/MS for quantitative analysis of small molecules

“…Furthermore, the possible in-source fragmentation in the case that the target analyte is a derivative of another intact biomarker molecule and natural isotope contribution might add another challenge to MS/MS (infusion or fl ow injection) only assays (Garg and Dasouki, 2006;Piraud et al, 2003). Consequently, LC-MS/MS has been increasingly explored in the confi rmatory analysis (second-tier test) of those 'diffi cult' biomarker molecules, resulting in a reduced false-positive rate (Janzen et al, 2007;Lacey et al, 2004;la Marca et al, 2007 and2008a;Matern et al, 2007;Minutti et al, 2004).…”