Comprehensive screening of urine samples for inborn errors of metabolism by electrospray tandem mass spectrometry

BACKGROUND: Detection of abnormal metabolites in urine is important for the diagnosis of many inborn errors of metabolism (IEM). Rapid, comprehensive screening methods are needed. METHODS: We used electrospray ionization tandem mass spectrometry in positive- and negative-ion modes to detect selected metabolites in urine. For positive-ion analysis, samples were dried and butylated, whereas for negative-ion analysis, samples were merely diluted with the mobile phase. Analysis was by direct injection with multiple reaction monitoring for 32 metabolites in positive mode (amino acids and acylcarnitines) and 30 metabolites in negative mode (organic acids). Run time was 2.1 min in each mode. RESULTS: Interbatch CVs ranged from 4.8% to 32%, enabling quantification of many metabolites. The procedure was applied to controls (278 and 120 in positive- and negative-ion mode, respectively) and 108 IEM individuals representing 37 different IEM. In 105 IEM individuals, representing 36 different IEM, concentrations of one or more diagnostic metabolites were above the 99th percentiles of the control values. CONCLUSIONS: The procedure is faster and less labor-intensive than conventional methods of testing for IEM by amino and organic acid profiling and has similar diagnostic sensitivity. The ability to include a greater range of metabolites offers the potential of a more comprehensive screening procedure.     

The screening of inborn errors of metabolism in sick Chinese infants by tandem mass spectrometry and gas chromatography/mass spectrometry

BackgroundAnalyses of amino acid/acylcarnitines in dried blood spots (DBS) and organic acids in urine are the primary tests for inborn errors of metabolism (IEMs). Automated tandem mass spectrometry (MS/MS) and gas chromatography/mass spectrometry (GC/MS) can rapidly and simultaneously detect numerous metabolic compounds with high precision and sensitivity.MethodsThree thousand four hundred and twenty-nine DBSs and 2781 urine samples were collected from our hospital patients with suspected IEMs, and analyzed for amino acid/acylcarnitines and organic acids by MS/MS and GC/MS, respectively. The results were used in a coincidental survey to determine the efficacy of these methods for the diagnosis of IEMs.ResultsNineteen different types of IEMs were detected in 121 affected cases (1.95% of 6210 samples). There were 66.12% amino acid disorders, 29.75% organic acid disorders and 4.13% with fatty acid oxidation disorders. Conclusions: the sick infants tested in this study had high prevalence rates of neonatal intrahepatic cholestasis, methylmalonic acidemia, hyperphenylalaninemia, tyrosinemia type I, and urea cycle disorders.ConclusionThe combined use of MS/MS and GC/MS is an appropriate tool for screening of IEMs in sick infants.     

Screening for inborn errors of metabolism using automated electrospray tandem mass spectrometry: Study in high-risk Indian population

Objectives:Tandem mass spectrometry is a major technological advance in the screening for inborn errors of metabolism. It has the advantage of sensitive and simultaneous multiple disease screening with minimal sample requirement. The diseases detected include aminoacidemias, fatty acid oxidation disorders, and organic acidemias.Design and methods:Using automated electrospray tandem mass spectrometry we screened 3550, clinically selected, symptomatic children for inborn errors of metabolism by analyzing amino acids and acylcarnitines in dried blood filter-paper samples.Results:Among these, 113 (3.2%) children were identified with a metabolic disorder: 61 (54%) patients had amino acid disorders, 47 (41.6%) had organic acidemias, and 5 (4.4%) children had disorders of fatty acid oxidation. The diagnoses were further confirmed through clinical symptoms, and other biochemical studies.Conclusions:These results show that inherited metabolic disorders are not rare in India, a rapidly developing country with a high birth rate and relatively frequent occurrence of consanguineous marriages.     

A primer on expanded newborn screening by tandem mass spectrometry

Newborn screening (NBS) for a wide variety of inborn errors of metabolism using the powerful tool of tandem mass spectrometry (MS/MS) is becoming widespread. This article provides the basic information that primary care clinicians need to help make rational decisions about urgent evaluation and appropriate follow-up of patients with abnormal expanded NBS tests. Also outlined are ways that the screening tests can be inaccurate. A normal MS/MS NBS result does not rule out the possibility of an inborn error of metabolism in an infant with acute illness, developmental abnormalities,or other clinically suggestive conditions. Primary care practitioners should identify the metabolic specialists in their area and develop a relationship with them to ensure clear communication when abnormal results arrive. Included in this article is a catalog of "thumbnail sketches" for reference by physicians when they receive an abnormal MS/MS NBS result.     

Application of tandem mass spectrometry to biochemical genetics and newborn screening

BACKGROUND: Tight regulation of blood glucose levels from patients suffering from diabetes mellitus can significantly reduce the complications associated with this disease. For this reason, elaborate research efforts have been devoted to the development of a glucose sensor for the continuous in vivo monitoring of glucose. Although the use of microdialysis as a sampling interface between the body and the biosensor is widely accepted, a major drawback of conventional microdialysis is the limited in vivo recovery. Here, ultraslow microdialysis is proposed in order to obtain (near) quantitative in vivo recoveries. To avoid, however, unacceptable long delay times, the need for a small and low dead volume measuring device was recognised. METHODS: A portable lightweight measuring device for continuous in vivo monitoring of glucose in subcutaneous tissue is presented. The measuring device consists of a miniaturised flow-through biosensor, connected to a microdialysis probe and a semi-vacuum pump. The biosensor is based on the amperometric detection of hydrogen peroxide after conversion of glucose by immobilised glucose oxidase. A portable potentiostat equipped with data logging is used for detection and registration. RESULTS: The device was validated for its accuracy, precision, linearity, sensitivity, selectivity and stability during ex vivo and in vivo experiments. The linearity was found to be up to 30 mmol/l with a limit of detection of 0.05 mmol/l. The precision, depending on the biosensor tested was found to be 2-4%. No contribution to the signal could be observed from several tested electroactive species. The accuracy was found to be well in accordance with the criteria set for methods of Self Monitoring of Blood Glucose for patients with diabetes mellitus. The biosensors could be used for up to 3 days in the continuous mode. In vivo monitoring of glucose in dialysate of subcutaneous sampled tissue during glucose tolerance tests in healthy volunteers demonstrated the potential of this measuring device. CONCLUSIONS: A portable lightweight measuring device is presented which can measure continuously glucose in vivo without excessive calibration steps. The performance characteristics determined justify the application of this measuring device for the in vivo monitoring of glucose in subcutaneous sampled interstitium of diabetic patients.     

Proficiency testing outcomes of 3-hydroxyisovalerylcarnitine measurements by tandem mass spectrometry in newborn screening

BackgroundThe use of tandem mass spectrometry (MS/MS) for the analysis of amino acids and acylcarnitines from dried-blood spots (DBS) has become routine practice in newborn screening laboratories. The Newborn Screening Quality Assurance Program (NSQAP) added 3-hydroxyisovalerylcarnitine (C5OH) into its routine quality control and proficiency testing (PT) DBS materials for MS/MS to assure the quality of C5OH screening. We report the results from NSQAP evaluations for C5OH-enriched DBS, and summarize participant screening practices based on their analytical methods.MethodsNSQAP prepared C5OH-enriched DBS materials for its participants. Laboratories reported quantitative and qualitative results. Bias plots of quantitative results were constructed using reported data and the results were sorted by an analytical method.ResultsNSQAP participants reported PT specimen 3964 as outside of normal limits for C5OH. The mean C5OH value for derivatized and non-derivatized methods was 2.80 and 2.67 μmol/l, respectively. Reported data from other specimens showed a similar trend in derivatized vs. non-derivatized assay results. Differences in C5OH quantitative values were observed among laboratories using different internal standards.ConclusionsC5OH MS/MS measurements in DBS assays varied by method and the choice of internal standards. The use of NSQAP's DBS materials allows harmonization of C5OH measurements by newborn screening laboratories worldwide.     

Expanded newborn screening of inherited metabolic disorders by tandem mass spectrometry: clinical and laboratory aspects

Newborn screening started in the 1960s for the purpose of identifying phenylketonuric patients to begin early intervention and to prevent mental retardation in these patients. Soon thereafter, screening programs expanded to include additional genetic disorders added individually one at a time. In the 1980s, tandem mass spectrometry (MS/MS) was introduced in clinical laboratories, and in the 1990s, the technique was used for newborn screening. Unlike measuring one analyte at a time, MS/MS allows measurement of >40 analytes, in a few minutes with the use of a single assay. Currently, MS/MS is being used for the identification of several amino acid, organic acid and fatty acid disorders. Several states in the United States and many other countries are using MS/MS in newborn screening. However, there is a significant disparity among different newborn screening programs for disorders being screened by MS/MS and many other challenges are faced by the expanded newborn screening. It is anticipated that in the future the use of MS/MS in newborn screening will expand both at the analyte and geographic levels. Clinicians and laboratory scientists should become familiar with MS/MS, disorders being screened in their patients' population and the future of this emerging technology.     

Improving the uptake of newborn screening for endocrinopathies and inborn errors of metabolism: A quality improvement initiative

IntroductionNewborn Screening (NBS) is a globally recognized essential, preventive public health program for identifying life threatening and debilitating conditions.MethodThis single centre quality improvement (QI) initiative was taken to improve the newborn screening uptake from the existing rate of 70%–100% over a period of three months in a tertiary care facility of India. A QI team was constituted to evaluate the reasons for low uptake of NBS and introduce changes in the existing system of NBS.ResultsBaseline data collection along with process mapping, and fishbone analysis were done. Problems attributing to most of the missed cases of newborns were studied using Pareto chart. Total three plan-do-study-asct (PDSA) cycles were used to fix the identified problems in order to improve the rate of NBS. The results of PDSA cycles were discussed among the team members. Frequency and percentage was computed for the collected data, after coding and entering in excel sheet. .ConclusionThe QI project on NBS successfully improved the rate of uptake of newborns for NBS from baseline to 100%.     

Improved specificity of newborn screening for congenital adrenal hyperplasia by second-tier steroid profiling using tandem mass spectrometry

BACKGROUND: Newborn screening for congenital adrenal hyperplasia (CAH) involves measurement of 17alpha-hydroxyprogesterone (17-OHP), usually by immunoassay. Because this testing has been characterized by high false-positive rates, we developed a steroid profiling method that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure 17-OHP, androstenedione, and cortisol simultaneously in blood spots. METHODS: Whole blood was eluted from a 4.8-mm (3/16-inch) dried-blood spot by an aqueous solution containing the deuterium-labeled internal standard d(8)-17-OHP. 17-OHP, androstenedione, and cortisol were extracted into diethyl ether, which was subsequently evaporated and the residue dissolved in LC mobile phase. This extract was injected into a LC-MS/MS equipped with pneumatically assisted electrospray. The steroids were quantified in the selected-reaction monitoring mode by use of peak areas in reference to the stable-isotope-labeled internal standard. We analyzed 857 newborn blood spots, including 14 blood spots of confirmed CAH cases and 101 of false-positive cases by conventional screening. RESULTS: Intra- and interassay CVs for 17-OHP were 7.2-20% and 3.9-18%, respectively, at concentrations of 2, 30, and 50 microg/L. At a cutoff for 17-OHP of 12.5 microg/L and a cutoff of 3.75 for the sum of peak areas for 17-OHP and androstenedione divided by the peak area for cortisol, 86 of the 101 false-positive samples were within reference values by LC-MS/MS, whereas the 742 normal and 14 true-positive results obtained by conventional screening were correctly classified. CONCLUSION: Steroid profiling in blood spots can identify false-positive results obtained by conventional newborn screening for CAH.     

Comparison of amino acids and acylcarnitines assay methods used in newborn screening assays by tandem mass spectrometry

BackgroundThe analysis of amino acids (AA) and acylcarnitines (AC) by tandem mass spectrometry (MS/MS) is performed in newborn screening laboratories worldwide. While butyl esterification assays are routine, it is possible to detect AAs and ACs as their native free acids (underivatized). The Centers for Disease Control and Prevention's Newborn Screening Quality Assurance Program provides dried blood spot (DBS) quality control (QC) and proficiency testing (PT) programs for numerous MS/MS analytes. We describe empirical differences between derivatization and non-derivatization techniques for selected AAs and ACs.MethodsDBS materials were prepared at levels near, above and below mean domestic laboratory cut-offs, and distributed to program participants for MS/MS analysis. Laboratories reported quantitative and qualitative results. QC DBS materials were assayed in-house following established protocols.ResultMinor differences (< 15%) between quantitative values resulting from butyl esters and free acid techniques were observed for the majority of the analytes. Mass spectrometric response from underivatized dicarboxylic acid acylcarnitines was less intense than their butyl esters.ConclusionsThe use of underivatized techniques may also result in the inability to differentiate isobaric acylcarnitines. Laboratories should establish their own protocols by focusing on the decisions that identify test results requiring additional follow-up testing versus those that do not.     

Validation of the phenylalanine/tyrosine ratio determined by tandem mass spectrometry: sensitive newborn screening for phenylketonuria.

The efficacy of the tandem mass spectrometry as a tool for the newborn screening of phenylketonuria, an inherited metabolic disorder, was investigated. Precision, reproducibility, selectivity and sensitivity were validated for phenylalanine and tyrosine measurements from dried blood spots. Bland-Altman plots were used to assess the agreement with conventional methods like fluorometry and ion exchange chromatography. The utility of the phenylalanine/tyrosine ratio for discrimination between mild hyperphenylalaninemia and classical types of phenylketonuria was investigated. Depending on concentration levels of phenylalanine and tyrosine the within-run and between-run assay variability ranged between 4.2% and 12.7%. Higher recoveries and a lower detection limit were found for the mass spectrometric method when compared to the fluorometric method. Pearson correlation coefficients of 0.91 for tandem mass spectrometry vs. fluorometric method, as well as 0.95 for tandem mass spectrometry vs. ion exchange chromatography were calculated. The closest agreement between methods was observed between tandem mass spectrometry and ion exchange chromatography. The results demonstrate a high efficacy of the tandem mass spectrometric method for quantitative determination of phenylalanine and tyrosine from dried blood spots. The phenylalanine/tyrosine ratio is crucial to improve the specificity and positive predictive value for the diagnosis of classical phenylketonuria.     

Newborn screening for hepatorenal tyrosinemia-I by tandem mass spectrometry using pooled samples: A four-year summary by the New England newborn screening program

ObjectiveThe objective of this study is to develop an isotope dilution liquid chromatography tandem mass spectrometry assay to screen for hepatorenal tyrosinemia (HT) from newborn filter paper samples using pooled extracts to increase high throughput screening.Design and methodsSuccinylacetone (SUAC), the marker for HT, was extracted from dried blood spots with the formation of the hydrazone derivative of SUAC; up to eight sample extracts were pooled and the SUAC-derivative was analyzed by mass spectrometry methods with an injection-to-injection time of one minute. If any pooled sample extract screened positive, then the samples comprising the pooled sample were assayed individually.ResultsTwo newborn infants were identified with high levels of SUAC (7 & 23 μM) and later confirmed to have HT. Three older children whose initial filter paper samples were taken at 195 days to 614 days of age with elevated SUAC (range 4.9–5 μM) were identified; one of the three had clinical signs of HT and was placed on treatment (diagnosis of the other two are unavailable).ConclusionMS/MS analysis of pooled dried blood sample extracts permits sensitive, reduced instrumental analytical time and increase high throughput screening for HT.