, Melissa Yssel, MB ChB, FC Path(SA) Chem 139, and Wendy M. Zakowicz, BS 79 Purpose: To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry through a worldwide collaborative effort. Methods: Cumulative percentiles of amino acids and acylcarnitines in dried blood spots of approximately 25-30 million normal newborns and 10,742 deidentified true positive cases are compared to assign clinical significance, which is achieved when the median of a disorder range is, and usually markedly outside, either the 99th or the 1st percentile of the normal population. The cutoff target ranges of analytes and ratios are then defined as the interval between selected percentiles of the two populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity taking all available factors into consideration.

Tandem mass spectrometry (MS/MS) has become a leading technology used in clinical chemistry and has shown to be particularly sensitive and specific when used in newborn screening (NBS) tests. The success of tandem mass spectrometry is due to important advances in hardware, software and clinical applications during the last 25 years. MS/MS permits a very rapid measurement of many metabolites in different biological specimens by using filter paper spots or directly on biological fluids. Its use in NBS give us the chance to identify possible treatable metabolic disorders even when asymptomatic and the benefits gained by this type of screening is now recognized worldwide. Today the use of MS/MS for second-tier tests and confirmatory testing is promising especially in the early detection of new disorders such as some lysosomal storage disorders, ADA and PNP SCIDs, X-adrenoleucodistrophy (X-ALD), Wilson disease, guanidinoacetate methyltransferase deficiency (GAMT), and Duchenne muscular dystrophy. The new challenge for the future will be reducing the false positive rate by using second-tier tests, avoiding false negative results by using new specific biomarkers and introducing new treatable disorders in NBS programs.

Inborn errors of metabolism are genetic disorders due to impaired activity of enzymes, transporters, or cofactors resulting in accumulation of abnormal metabolites proximal to the metabolic block, lack of essential products or accumulation of by-products. Many of these disorders have serious clinical consequences for affected neonates, and an early diagnosis allows presymptomatic treatment which can prevent severe permanent sequelae and in some cases death. Expanded newborn screening for these diseases is a promising field of targeted metabolomics. Here we report the application, between 2007 and 2014, of this approach to the identification of newborns in southern Italy at risk of developing a potentially fatal disease. The analysis of amino acids and acylcarnitines in dried blood spots by tandem mass spectrometry revealed 24 affected newborns among 45,466 infants evaluated between 48 and 72 hours of life (overall incidence: 1 : 1894). Diagnoses of newborns with elevated metabolites were confirmed by gas chromatography-mass spectrometry, biochemical studies, and genetic analysis. Five infants were diagnosed with medium-chain acyl CoA dehydrogenase deficiency, 1 with methylmalonic acidemia with homocystinuria type CblC, 2 with isolated methylmalonic acidemia, 1 with propionic acidemia, 1 with isovaleric academia, 1 with isobutyryl-CoA dehydrogenase deficiency, 1 with beta ketothiolase deficiency, 1 with short branched chain amino acid deficiency, 1 with 3-methlycrotonyl-CoA carboxylase deficiency, 1 with formimino-transferase cyclodeaminase deficiency, and 1 with cystathionine-beta-synthase deficiency. Seven cases of maternal vitamin B12 deficiency and 1 case of maternal carnitine uptake deficiency were detected. This study supports the widespread application of metabolomic-based newborn screening for these genetic diseases.

Enzyme replacement therapy is currently the only approved treatment for Pompe disease, due to acid α-glucosidase deficiency. Clinical efficacy of this approach is variable, and more effective therapies are needed. We showed in preclinical studies that chaperones stabilize the recombinant enzyme used for enzyme replacement therapy. Here, we evaluated the effects of a combination of enzyme therapy and a chaperone on α-glucosidase activity in Pompe disease patients. α-Glucosidase activity was analyzed by tandem-mass spectrometry in dried blood spots from patients treated with enzyme replacement therapy, either alone or in combination with the chaperone N-butyldeoxynojirimycin given at the time of the enzyme infusion. Thirteen patients with different presentations (3 infantile-onset, 10 late-onset) were enrolled. In 11 patients, the combination treatment resulted in α-glucosidase activities greater than 1.85-fold the activities with enzyme replacement therapy alone. In the whole patient population, α-glucosidase activity was significantly increased at 12 hours (2.19-fold, P = 0.002), 24 hours (6.07-fold, P = 0.001), and 36 hours (3.95-fold, P = 0.003). The areas under the curve were also significantly increased (6.78-fold, P = 0.002). These results suggest improved stability of recombinant α-glucosidase in blood in the presence of the chaperone.

Background: Adenosine deaminase (ADA)-severe combined immunodeficiency (SCID) is caused by genetic variants that disrupt the function of ADA. In its early-onset form, it is rapidly fatal to infants. Delayed or late-onset ADA-SCID is characterized by insidious progressive immunodeficiency that leads to permanent organ damage or death. Quantification of T-cell receptor excision circles (TRECs) or tandem mass spectrometry (tandem-MS) analysis of dried blood spots (DBSs) collected at birth can identify newborns with early-onset ADA-SCID and are used in screening programs. However, it is not clear whether these analyses can identify newborns who will have delayed or late-onset ADA-SCID before symptoms appear. Objective: We performed a retrospective study to evaluate whether tandem-MS and quantitative TREC analyses of DBSs could identify newborns who had delayed-onset ADA-SCID later in life. Methods: We tested stored DBSs collected at birth from 3 patients with delayed-onset ADA-SCID using tandem-MS (PCT EP2010/070517) to evaluate levels of adenosine and 29-deoxyadenosine and real-time PCR to quantify TREC levels. We also analyzed DBSs from 3 newborns with early-onset ADA-SCID and 2 healthy newborn carriers of ADA deficiency. Results: The DBSs taken at birth from the 3 patients with delayed-onset ADA-SCID had adenosine levels of 10, 25,

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The expansion of national newborn screening (NBS) programmes has provided significant benefits in the diagnosis and early treatment of several rare, heritable conditions, preventing adverse health outcomes for most affected infants. New technological developments have enabled the implementation of testing panel covering over 50 disorders. Consequently, the increment of false positive rate has led to a high number of healthy infants recalled for expensive and often invasive additional testing, opening a debate about the harm-benefit ratio of the expanded newborn screening. The false-positive rate represents a challenge for healthcare providers working in NBS systems. Here, we give an overview on the most commonly used strategies for decreasing the adverse effects due to inconclusive screening results. The focus is on NBS performance improvement through the implementation of analytical methods, the application of new and more informative biomarkers, and by using post-analytical interpretive tools. These strategies, used as part of the NBS process, can to enhance the positive predictive value of the test and reduce the parental anxiety and healthcare costs related to the unnecessary tests and procedures.

a b s t r a c tErtapenem (Invanz ® ) is a newly developed carbapenem ␤-lactam antimicrobial agent. The drug usage in pediatric age needs an accurate drug monitoring for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to measure ertapenem concentration during treatment. The analysis was performed by UPLC-MS/MS operating in multiple reaction monitoring (MRM) mode. The calibration curve in matrix was linear in the concentration range of 0.5-100 mg/L with correlation coefficient value higher than 0.997. Performance parameters of this method like lower limit of detection (LLOD, 0.2 mg/L), lower limit of quantification (LLOQ, 0.5 mg/L), matrix effect (20%), intra-and inter-day imprecision (CV within than 15%) and accuracy (between 94 and 155%) of drug concentrations have been evaluated. The drug stability at different temperatures was tested for one month, to evaluate the risks of sample delivery at different climatic conditions.The reported method allows now ertapenem analysis and offers many advantages for patients including the possibility of collecting samples at home.This new assay is both precise and accurate and is especially suitable for therapeutic drug monitoring and pharmacokinetic studies in neonates in whom obtaining larger blood samples is not convenient or possible.