Development of Strategies to Decrease False Positive Results in Newborn Screening.

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.

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.

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).

Heptadecanoylcarnitine (C17) a novel candidate biomarker for newborn screening of propionic and methylmalonic acidemias.

Background: 3-Hydroxypalmitoleoyl-carnitine (C16:1-OH) has recently been reported to be elevated in acylcarnitine profiles of patients with propionic acidemia (PA) or methylmalonic acidemia (MMA) during expanded newborn screening (NBS). High levels of C16:1-OH, combined with other hydroxylated long chain acylcarnitines are related to long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and trifunctional protein (TFP) deficiency.

Methods: The acylcarnitine profile of two LCHADD patients was evaluated using liquid chromatography-tandem mass spectrometric method.

Expanded newborn screening by mass spectrometry: New tests, future perspectives.

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.

Therapeutic drug monitoring of carbamazepine and its metabolite in children from dried blood spots using liquid chromatography and tandem mass spectrometry.

Carbamazepine (CBZ) is a first-line drug for the treatment of different forms of epilepsy and the first choice drug for trigeminal neuralgia. CBZ is metabolized in the liver by oxidation into carbamazepine-10,11-epoxide (CBZE), its major metabolite which is equipotent and known to contribute to the pharmacological activity of CBZ. The aim of the present study was to develop and validate a reliable, selective and sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of CBZ and its active metabolite in dried blood spots (DBS).

Targeted metabolomics in the expanded newborn screening for inborn errors of metabolism.

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.

Dried blood spot assay for the quantification of phenytoin using Liquid Chromatography-Mass Spectrometry.

Phenytoin (PHT) is one of the most commonly used anticonvulsant drugs for the treatment of epilepsy and bipolar disorders. The large amount of plasma required by conventional methods for drug quantification makes mass spectrometry combined with dried blood spot (DBS) sampling crucial for pediatric patients where therapeutic drug monitoring or pharmacokinetic studies may be difficult to realize. DBS represents a new convenient sampling support requiring minimally invasive blood drawing and providing long-term stability of samples and less expensive shipment and storage.

A chaperone enhances blood α-glucosidase activity in Pompe disease patients treated with enzyme replacement therapy.

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.

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

Background: Purine nucleoside phosphorylase (PNP) deficiency is a rare form of autosomal recessive combined primary immunodeficiency caused by a enzyme defect leading to the accumulation of inosine, 2'-deoxy-inosine (dIno), guanosine, and 2'-deoxy-guanosine (dGuo) in all cells, especially lymphocytes. Treatments are available and curative for PNP deficiency, but their efficacy depends on the early approach. PNP-combined immunodeficiency complies with the criteria for inclusion in a newborn screening program.

Screening of lysosomal storage disorders: application of the online trapping-and-cleanup liquid chromatography/mass spectrometry method for mucopolysaccharidosis I.

In recent years, new treatments have become available to treat some lysosomal storage disorders (LSDs) and many studies suggest that there is a benefit with starting therapy early. Newborn screening should detect diseases early enough for prompt treatment. Some countries include additional conditions, such as some LSDs, into their newborn screening panels.

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

Severe 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.

A rapid liquid chromatography tandem mass spectrometry-based method for measuring propranolol on dried blood spots.

Propranolol, a non-selective beta blocker drug, is used in young infants and newborns for treating several heart diseases; its pharmacokinetics has been extensively evaluated in adult patients using extrapolation to treat pediatric population. The purpose of the present study was to develop and validate a method to measure propranolol levels in dried blood spots. The analysis was performed by using liquid chromatography/tandem mass spectrometry operating in multiple reaction monitoring mode.

Tandem mass spectrometry, but not T-cell receptor excision circle analysis, identifies newborns with late-onset adenosine deaminase deficiency.

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.

Rapid and sensitive LC-MS/MS method for the analysis of antibiotic linezolid on dried blood spot.

Linezolid is a new drug from the oxazolidinone class of antibiotics used against mycobacteria and multi-drug resistant (MDR) Gram-positive bacterial infections, which may are also glycopeptide-resistant. 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 determinate linezolid levels during treatment.

Reversal of metabolic and neurological symptoms of phenylketonuric mice treated with a PAH containing helper-dependent adenoviral vector.

Phenylketonuria (PKU) is one of the most common inborn errors of metabolism and is due to a deficit of phenylalanine hydroxylase, the enzyme that converts phenylalanine (Phe) into tyrosine (Tyr). The resultant hyperphenylalaninemia (HPA) leads to severe neurological impairment, whose pathogenesis has not been entirely elucidated. Treatment of PKU consists essentially in lifelong protein restriction and, in mild cases, in tetrahydrobiopterin supplementation.

Development of an UPLC-MS/MS method for the determination of antibiotic ertapenem on dried blood spots.

Ertapenem (Invanz) is a newly developed carbapenem β-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.

Quantitative liquid chromatography coupled with tandem mass spectrometry analysis of urinary acylglycines: application to the diagnosis of inborn errors of metabolism.

The analysis of urinary acylglycines is an important biochemical tool for the diagnosis of many organic acidemias and mitochondrial fatty acid β-oxidation defects. A new rapid analytical method has been developed for quantification of acylglycines in urine by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The method requires a simple sample preparation avoiding derivatization.

The first case of mitochondrial acetoacetyl-CoA thiolase deficiency identified by expanded newborn metabolic screening in Italy: the importance of an integrated diagnostic approach.

A pilot expanded newborn screening programme to detect inherited metabolic disorders by means of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) began in the Campania region, southern Italy, in 2007. By October 2009, >8,800 dried blood samples on filter paper from 11 hospitals had been screened. Within this screening programme, we identified a case of mitochondrial acetoacetyl-coenzyme A (CoA) thiolase deficiency [β-ketothiolase (β-KT) deficiency] by analysing the acylcarnitine profile from a dried blood spot with LC-MS/MS.