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Synopses of Selected Papers Presented at the Tandem Mass Spectrometry for Metabolic Disease Screening Among Newborns Workshop, San Antonio, Texas, June 2000

Scientists from all operating tandem mass spectrometry (MS/MS) newborn screening laboratories and programs in the United States public health, academic, and private and two international experts (Rodney J. Pollitt, Ph.D., United Kingdom and Bridget Wilcken, M.B., Ch.B., Australia) were invited as discussants for the workshop held in June 2000. The discussant group included the pioneers of MS/MS applications in newborn screening, Charles R. Roe, M.D.; Donald H. Chace, Ph.D.; David S. Millington, Ph.D.; and Edwin W. Naylor, Ph.D., as well as scientists from the two companies that manufacture MS/MS equipment used for newborn screening. The workshop was a joint effort that involved cooperation among public health agencies, academic institutions, and the private sector with shared interest in enhancing the use and quality of MS/MS technology. Approximately 50 observers from newborn screening programs in the United States with future expectations for using MS/MS also attended the workshop.

Each invited discussant was asked to provide a 1-page summary relating their experiences with MS/MS for any of the applicable parameters (e.g., setting-up and operating MS/MS, selection of cutoffs, associated follow-up of infants with identified disorders, medical confirmation of disorders, and treatment of disorders). Summaries were submitted of presentations given at the plenary session and breakout group meetings. Invited discussants were also asked to provide a list of what they believed were the 510 most important questions, in a priority order, regarding the application and needs for MS/MS in screening, confirmation, quality assurance, follow-up, and treatment of metabolic disorders among newborns. These presentations, summaries, and lists of questions were used to guide breakout groups in their deliberations and discussions for the development of work group proposals for MS/MS in newborn screening. Selected synopses of summary papers that represent the context of the workshop are presented here.


Testing Newborn Specimens by Tandem Mass Spectrometry: The First 16 Months' Experience in the New England Program Thomas H. Zytkovicz, Ph.D.; Donna Johnson; Denise Rojas, M.P.H.; Eileen Fitzgerald; New England Newborn Screening Program, Jamaica Plain, Massachusetts

On February 1, 1999, the New England Newborn Screening Program began using tandem mass spectrometry (MS/MS) to test newborns for 23 disorders, including 9 amino acid disorders, 7 organic aciduria disorders, and 7 fatty acid oxidation disorders. However, before using MS/MS, we validated the experimental method.* MS/MS instrument bias was determined by comparing test results with preexisting analytical methods (e.g., high-pressure liquid chromatography and bacterial inhibition assay). Blood controls were prepared by fortifying samples with amino acids and acylcarnitines and sending these samples to other laboratories for MS/MS analysis of the markers. These interlaboratory results were used to compare our laboratory's performance with that of established laboratories. Approximately 170,400 newborns were tested for phenylketonuria, maple syrup urine disease, and homocystinuria. MS/MS identified six infants with phenylketonuria, nine with hyperphenylalaninemia, one with maple syrup urine disease, and three infants with homocystinuria/hypermethionemia. Approximately 100,000 newborns were tested for 20 other disorders. MS/MS tentatively identified seven infants with medium-chain acyl-CoA dehydrogenase; four with short-chain acyl-CoA dehydrogenase; two with phenolic acid; and one each with methylcrotonyl-CoA carboxylase; carnitine palmitoyl transferase, type II; and very-longchain acyl-CoA dehydrogenase. The majority of these infants are undergoing DNA (deoxyribonucleic acid), urine, or in vitro testing to confirm MS/MS diagnoses.

Comprehensive, High-Quality Analytical Approach to Newborn Screening Using Tandem Mass Spectrometry Donald H. Chace, Ph.D.; Neo Gen Screening, Inc., Bridgeville, Pennsylvania

Because MS/MS technology can be used for screening multiple disorders in a single analysis, low-prevalence disorders can be included with higher prevalence analytes without a substantial additional cost. However, MS/MS screening results require interpretation by experienced metabolic specialists and confirmation by other analytical or diagnostic techniques. MS/MS uses stable isotope internal standards as an essential component of the method, and therefore, it is precise and accurate in controlled tests. Nevertheless, blood volume from filter paper blood-spot specimens can be inaccurate and reduce the reproducibility of MS/MS that is observed in analyses of liquid specimens (e.g., serum or plasma). Ratios of >2 components improve diagnostic accuracy and reproducibility that otherwise would be lost by using filter paper blood-spot specimens. As this emerging technology develops, sample preparation, analyses, data processing, and interpretations will be modified. New assays will expand the current panel of MS/MS-detectable disorders or provide a source for confirmation. Further, as genetic testing expands to include additional molecular approaches, complementation of these methods will provide comprehensive analyses that will better serve public health needs.

Introduction of Tandem Mass Spectrometry Into the Newborn Screening Environment Michael R. Morris, Ph.D.; Micromass United Kingdom Ltd., Manchester, United Kingdom

Personnel training and laboratory support can resolve certain problems related to introducing tandem mass spectrometry for newborn screening, but program managers must develop their own experiences before determining that MS/MS is a stable tool to be used in routine newborn screening. Although a detailed knowledge of physics and instrument operation is unnecessary, program managers and laboratorians should understand basic operational details. High-throughput screening of extracted blood samples is an exacting test, and certain critical factors must be considered to maintain optimum instrument performance, including the following:

  • Sample preparation. If samples are prepared in a substandard manner, the validity of the assay becomes questionable. Addition of internal standard materials at the beginning of the extraction procedure allows monitoring of the extraction and derivatization as well as instrument performance. Quality of solvents, standards, and other materials used in sample preparation procedures must be assessed for suitability.
  • Instrument sensitivity. Using a benchmark solution to visually check instrument performance at the start of a day's work can identify problems in a timely manner. Injection of a standard sample that can be monitored in real time can give the experienced user information regarding the status of the flow path and injection system, the cleanliness of the ion source, and the status of the mass calibration. Users become adept at rapidly drawing conclusions on the basis of the arrival time of the sample after injection, the location of the individual peaks on the mass scale, the instrument resolution, and the absolute and relative intensities of the peaks.
  • Sample inlet system blockages. Investigative samples contain a substantial number of organic-soluble compounds and have the potential to introduce particulate matter. A logical assessment of the liquid-flow path and regular monitoring of the back-pressure of the liquid chromatography pump will increase efficiency.
  • Use of operational qualification and quality-control samples. Ideally, quality-control samples should be used to monitor the performance of the assay for all compounds of interest. Practically, monitoring an individual compound from each class of analytes being measured might be sufficient to prove analytical effectiveness. Also, tests should be run to ensure that particular internal standards are present at the correct concentration.

Laboratories that have played key roles in introducing MS/MS into newborn screening have developed cross-checks to monitor assay performance and provide analytical safeguards. In addition, interlaboratory communication that focuses on emerging problems and possible solutions is strongly recommended.

Selection of Reporting Cutoffs in Newborn Screening: Patient-to-Normal Ratio Joerg N. Pirl, Ph.D.; Rong Shao, M.D.; Michael Petros, M.S., M.P.H.; Illinois Department of Public Health, Chicago, Illinois

The selection process for reporting cutoffs is based on statistical evaluations of historical patient data, which might change as methods and procedures are updated, thus producing a substantial number of false-positive results. Because test results are method-dependent, reference ranges or normal values are needed for interpretation. If the patient results are expressed relative to that normal value, the resulting patient-to-normal ratio (PNR) is a unitless number that is a measure of the deviation of the patient result from that of the normal population. When normal values are calculated from within-run data, uniform variations associated with matrix, calibration, recovery, accuracy, and instrument will cancel. In a newborn screening laboratory, approximately 95% of samples received are from patients with normal levels, and PNRs derived from patients analyzed under approximately identical circumstances are method-independent and, thus, interlaboratory comparable.**

We compared rapid-flow analysis (RFA) and MS/MS phenylalanine (phe) results from approximately 14,000 neonatal dried blood spots. Included were 267 patients with levels above our RFA cutoff of 4.0 mg/dL and 45 patients with confirmed phenylketonuria or hyperphenylalaninemia. For newborns with normal levels, concentrations of phe were usually distributed around the mean. When the results were arranged in ascending order, the result of the median patient agreed closely with the mean of the median 68% (mean ± 1 standard deviation [SD]) and 95% (mean ± 2 SD) of the population. This agreement was true also for ratios of phe to selected other amino acids. The median deviated by <1% from the 68% and 95% population mean, and the concentration of phe as well as the ratio of phe to the other amino acids was linearly related to the median 60% of the normal patients with a slope near zero; for phe, the slope was 0.00087 mg/dL/patient. That is, when results were sorted numerically, this group of patients had the same or nearly the same results. For any given routine run (i.e., MS/MS or RFA, with a minimum of one 96-well microplate) the result of the median patient (patients arranged in ascending order) differed <3% from the mean of the central 68% and <5% from the central 95% mean. MS/MS, in light of its high analyte specificity, produced a lower phe concentration than RFA but did not substantially improve the predictability for disease-correlation coefficient, MS/MS versus RFA >0.9. RFA positive predictive value was 18.2%. To examine internal metabolite ratios and to minimize effects from specimen quality or feeding, in addition to phe, we also analyzed these patients by MS/MS for leucine (leu), methionine (met), tyrosine (tyr), alanine (ala), valine (val), and serine (ser).

PNR was calculated for phe, phe/leu, phe/met, phe/tyr, phe/ala, phe/val, and phe/ser by dividing the results of the patients by the median result for that parameter, with results sorted numerically. The final output presented the PNR values for phe and those of the internal metabolic ratios in tabular form. PNR-phe/tyr of <2.0 was associated with normalcy. The lowest PNR-phe/tyr in hyperphenylalaninemia patients was 2.36 and 4.62 in cases of classic phenylketonuria. The positive predictive value was 83.8% if PNR for all internal ratios was considered and 38.4% on the basis of PNR for phe/tyr only. The negative predictive value was 100% in all cases.

Implementation of Tandem Mass Spectrometry in Wisconsin's Newborn Screening Program Gary L. Hoffman; Thomas Litsheim; Ronald H. Laessig, Ph.D.; Wisconsin State Laboratory of Hygiene, Madison, Wisconsin

After we acquired a tandem mass spectrometry (MS/MS) instrument to measure acylcarnitines in dried blood specimens, we devoted approximately 1 month to learning its operation. This learning period consisted of a) one, 2-day session with the service engineer during the setup reviewing instrument calibration and routine maintenance requirements; b) two, 3-day sessions with instrument manufacturer's application specialists to establish sample preparation, method calibration, and data reduction; c) 1 week independently developing a comfort level with instrument operation; and d) 1 week at a training course at the manufacturer's facility. This training enabled us to begin pilot testing.

One of the goals of the pilot testing phase was to collect acylcarnitine data to establish reporting profiles for fatty acid oxidation and organic aciduria disorders. The first step was a literature review of the acylcarnitines associated with each of the 14 fatty acid oxidation and organic aciduria disorders mandated by the Wisconsin Department of Health's Newborn Screening Advisory Committee. Although published literature does not agree on the acylcarnitine profile for each disorder, we established a composite profile for each one. After collecting 5,000 random observations for each acylcarnitine, we established an abnormal reporting level at the mean concentrations plus four standard deviations. We further adjusted these abnormal reporting levels as follows: a) comparing them against those already published by other laboratories using MS/MS to identify newborns with these 14 metabolic disorders, and b) consulting with an experienced metabolic expert in the clinical diagnosis of these disorders. For the majority of acylcarnitines, the four standard deviation level was maintained, although certain adjustments were made that increased the cutoff of some acylcarnitines to >5 standard deviations from the mean. In addition to acylcarnitine levels, we added multiple concentration ratio-based criteria to the abnormal profiles for certain disorders. Using this preliminary criteria, we referred 13 babies for confirmatory testing from the approximately 50,000 specimens screened during the pilot study. Five (i.e., four medium-chain acyl-CoA dehydrogenase and one short-chain acyl-CoA dehydrogenase disorders) were confirmed.

A secondary goal of the pilot study was to determine how the MS/MS technology could be incorporated into the routine newborn screening operation. A primary focus was on the ability of the MS/MS instrumentation to process the specimens in a timely manner (i.e., <24 hours after receipt) so delay in reporting all results would be minimal. With modifications, we reduced sample preparation time to <10 minutes after punching*** the sample from the blood-spot specimen. Because the instruments' throughput is 1 sample/1.5 minutes and it can operate overnight unattended, the system could handle 500 samples/day. On the basis of the success of the pilot testing phase, which established abnormal reporting levels and instrument reliability, we began routine testing for 14 metabolic disorders by MS/MS in April 2000.


Incidence and Follow-Up Evaluation of Metabolic Disorders Detected by Newborn Screening in North Carolina Using Tandem Mass Spectrometry Joseph Muenzer, M.D.; Dianne M. Frazier, Ph.D.; Shawn E. McCandless, M.D.; University of North Carolina, Chapel Hill, North Carolina; Elizabeth G. Moore, M.S.W.; Susan D. Weavil; Shu H. Chaing, Ph.D.; Department of Health and Human Services, Raleigh, North Carolina; David S. Millington, Ph.D.; Duke University Medical Center, Research Triangle Park, North Carolina

Since August 1997, the North Carolina Newborn Screening Program has been screening all newborn infants by using tandem mass spectrometry (MS/MS) for selected amino acid, organic acid, and fatty acid oxidation disorders. Initially, a statewide pilot study lasting 20 months was conducted, and on the basis of that pilot study, the North Carolina Newborn Screening Program incorporated MS/MS screening into the state newborn screening panel. During the pilot study, Neo Gen Screening, Inc., (Bridgeville, Pennsylvania) screened 194,384 newborns (214,634 specimens) and identified 259 (0.13%) samples as abnormal. Metabolic disorders were confirmed for 31 infants; results indicated that 14 children had medium-chain acyl-CoA dehydrogenase deficiency; 1, long-chain fatty acid oxidation disorder; 6, hyperphenylalaninemia; 1, hypermethionemia; 3, citrullinemia; 1, argininosuccinic aciduria; and 5, organic aciduria disorders.

During a subsequent phase of implementation (April 1999June 2000), the North Carolina Newborn Screening Laboratory screened 131,776 newborns (147,286 specimens). Initial cutoffs resulted in false-positive detection rates of >1.9%, but revised cutoffs resulted in a <0.85% false-positive rate. Metabolic disorders were confirmed for 27 infants; results indicated that 10 children had medium-chain acyl-CoA dehydrogenase deficiency; 1, short-chain acyl-CoA dehydrogenase deficiency; 9, hyper-phenylalaninemia; and 7, organic aciduria disorders. Since MS/MS screening began in North Carolina, disorders among three infants (one with late-onset methylmalonic acidemia and two with glutaric acidemia, type I) were missed by MS/MS screening, but the disorders were diagnosed clinically before age 1 year for all three infants.

Patient follow-up in North Carolina is coordinated by the Division of Genetics and Metabolism, Department of Pediatrics, University of North Carolina at Chapel Hill (UNC). A two-tier follow-up approach has been used with abnormal MS/MS screening results. When the MS/MS results indicate an inborn error of metabolism, the screening laboratory notifies UNC by telephone and facsimile. UNC staff then contact the local health-care provider by using information provided on the newborn screening card. Follow-up evaluation and recommendations for additional testing, on the basis of the MS/MS laboratory findings, are made by UNC personnel so the suspected diagnosis can be confirmed, the family counseled, and treatment initiated. Initial MS/MS screening results that are borderline (i.e., a possible inborn error of metabolism) require repeat testing. If a second specimen indicates abnormal levels, UNC staff coordinate additional testing (i.e., for abnormal amino acids, a plasma amino acid analysis is run; for an abnormal acylcarnitine profile, plasma acylcarnitine profile and urinary organic acid analyses are run).

All infants in North Carolina with medium-chain acyl-CoA dehydrogenase deficiency detected by newborn screening are treated with carnitine and are maintained on breast milk or infant formula with no fat restriction to age 1 year. Parents are instructed to avoid prolonged fasting and to use a glucometer to monitor blood glucose levels when they are concerned regarding the child's oral intake or clinical status. No deaths, significant hypoglycemic events, or seizures have occurred among the infants with medium-chain acyl-CoA dehydrogenase deficiency identified by newborn screening. Screening by MS/MS, with careful follow-up, can prevent the majority of deaths and serious sequelae of medium-chain acyl-CoA dehydrogenase deficiency during the first years of life. However, MS/MS is not entirely specific for this error of metabolism and detection of abnormal but nondiagnostic metabolites can occur. Since MS/MS analysis began in the North Carolina Newborn Screening Laboratory, borderline results have been common with propionyl-acylcarnitine, 3-hydroxy-isovalerylcarnitine (C5-OH), and tyrosine. Adjustment of cutoff levels has reduced the false-positive rate, but additional adjustment might be needed on the basis of follow-up evaluation of the borderline results. Close coordination between the MS/MS screening laboratory and the metabolic clinic/biochemical geneticists is needed to determine screening parameters, adjust the cutoff levels to reduce false-positive and false-negative results, and facilitate clinical follow-up.

Notification Experience with Newborn Disorders Detected by Tandem Mass Spectrometry in the Early Experience of the New England Program George F. Grady, M.D.; Thomas H. Zytkovicz, Ph.D.; Deborah Marsden, M.D.; Cecilia Larson, M.D.; Vivian Shih, M.D.; New England Newborn Screening Program, Jamaica Plain, Massachusetts

A series of protocols has emerged for notifying pediatricians when a newborn under their care is identified by tandem mass spectrometry (MS/MS) screening as having an out-of-range amino acid or acylcarnitine value. For the amino acids, phenylalanine (for phenylketonuria [PKU] or hyperphenylalaninemia [HPA]), leucine plus isoleucine (for maple syrup urine disease [MSUD]), and methionine (for homocystinuria [HCU]), the MS/MS analysis was performed for approximately 171,500 newborns. The net yield of confirmed (repeatedly positive) cases was similar to that during the prior era of bacterial inhibition assay (i.e., six PKU plus nine HPA, one MSUD, and one HCU plus two hypermethionemia). Persistence of biological confounders affected clinical interpretation of the phenylalanine, leucine, or methionine for 102 newborns, who required repeat specimens because of immaturity or systemic illness.

For approximately 109,400 newborns, the screening profile by MS/MS included separate recordings of tyrosine and the basic amino acids, ornithine, citrulline, and arginine. Testing repeat specimens from 24 infants with an initial elevation of tyrosine, and from 24 infants who had initial elevations of ornithine, citrulline, or arginine, yielded no confirmed cases of specific disorders that would have explained the elevations. Nevertheless, when physicians associated with our screening program staff contacted the infants' physicians regarding the elevation, our staff provided information regarding potential signs and symptoms and possible future interventions.

The 109,400 newborns were also screened by MS/MS for certain acylcarnitines that had side chains containing <18 carbons (e.g., C18), to detect fatty acid oxidation and organic aciduria disorders. For an additional 10,000 infants, only the medium-chain acylcarnitines were measured. Among those infants whose test results indicated fatty acid oxidation disorders, the medium-chain candidate group included 36 infants of whom a) two had an initial C8 of 910 µM (the parents were referred immediately to metabolic clinics); b) five had an initial C8 of 1.83.2 µM (parents were given the option of direct referral or awaiting results of C8 on a second specimen before deoxyribonucleic acid or urinary acylglycine analysis); and c) 29 had an initial C8 elevation of 0.50.8 µM. The cases in group a were homozygous for the A985G marker; group b was incompletely analyzed but includes ones with positive urinary samples or a single copy of A985G; and group c revealed no elevated C8 on repeat testing and the deoxyribonucleic acid assay was left to personal choice. Upon initial notification, all pediatricians were asked to verify feeding status of the infant and to advise parents to avoid gaps in feeding.

Fatty acid oxidation disorder detections also included four presumptive cases of short-chain acyl-CoA dehydrogenase deficiency that had initially elevated butyrylcarnitine, C4 (range: 2.63.7 µM), persisting on a repeat specimen or accompanied by the respective urine acylglycine, deoxyribonucleic acid, or in vitro markers. In 19 other newborns, initial elevations of C4 (>1.9 µM) did not persist (the notification protocol was analogous to the medium-chain protocol). The remaining four notifications included two with an initial elevation of C16, one of which was a fatal case of carnitine palmitoyl transferase, type 2; and two with elevated C14:1, one of which was confirmed as very long-chain acyl-CoA dehydrogenase deficiency.

Organic aciduria disorder detections included two propionic acidemias (among 23 notifications when propionylcarnitine, C3, was >8 µM) and one beta-methylcrotonyl CoA carboxylase deficiency (among 12 notifications of C5 OH >0.8 µM). Because early onset central nervous system problems were a greater concern for this disorder than for fatty acid oxidation disorder, notification protocols were more aggressive in spite of lower specificity of the initial elevated marker.

Newborn Screening for Medium-Chain Acyl-CoA Dehydrogenase Deficiency Sophia S. Wang, Ph.D.; National Center for Environmental Health, CDC, Atlanta, Georgia

Increasingly, programs are screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency among newborns, which has prompted implementation of tandem mass spectrometry (MS/MS) technology for such testing. If newborn screening programs conduct systematic follow-up and collect data beyond diagnosis, they can serve as models to be used in evaluating programs, determining clinical validity and utility, and providing necessary population-based data. To ensure and evaluate current programs, the Institute of Medicine's core functions of public health (i.e., assessment, policy development, and assurance and evaluation) should serve as the evaluation framework.**** Program assurance and evaluation requires the collection of key follow-up data beyond the newborn period to track receipt of services and prevention of adverse outcomes. Essential data include short- and long-term process and outcome measures. Key short-term measures include the percentage of live-born infants adequately screened and the timeliness of diagnoses and treatment; essential long-term measures include assessment of adverse health outcomes beyond the newborn period. These data are also essential for developing future programs. Collection of follow-up data also facilitates assessment of key test parameters (i.e., clinical validity and utility),***** which provide information regarding testing accuracy and utility. Clinical validity is defined as how well the test predicts the phenotype; clinical utility measures the benefits and risks of early detection for those persons among whom disease is detected. Lastly, systematic collection of key follow-up parameters can provide needed population-based data regarding these rare disorders. Data regarding incidence, prevalence, and clinical outcomes can be determined for the populations being tested and contribute to the elucidation of the natural history of MCAD deficiency. Furthermore, the penetrance of the mutations associated with MCAD deficiency can be investigated. On the basis of rates of heterozygosity and under Hardy-Weinberg conditions, more MCAD deficiency cases are expected than are currently observed, leading to a substantial number of infants with asymptomatic MCAD deficiency and uncertainty as to who will manifest symptoms and who will remain asymptomatic. Although diagnosing MCAD deficiency among children is the primary goal of current newborn screening programs, including systematic collection of key follow-up data is vital to ensuring optimal functioning and utility of programs.

Grouping Metabolic Disorders Detected by Tandem Mass Spectrometry to Maximize Political Impact on Legislators and Policymakers William J. Rhead, M.D., Ph.D.; Medical College of Wisconsin, Elm Grove, Wisconsin

Effectively presenting tandem mass spectrometry (MS/MS) and universal newborn screening to legislators and policymakers is an essential preliminary step that differs from actually implementing expanded programs. When seeking legislative or administrative approval at the state or territorial level, presenting the screened disorders to maximize their comprehensibility for persons without scientific or medical backgrounds is useful. Thus, medium-chain acyl-CoA dehydrogenase disorder, long-chain hydroxy acyl-CoA dehydrogenase deficiency, and related hypoglycemic disorders could be grouped and presented under a "low blood sugar" or "sudden infant death syndrome-like" category. Disorders producing encephalopathy (e.g., hyperammonemias and maple syrup urine disease) could be grouped together in a "coma" category; and 3-methylcrotonyl carboxylase deficiency and propionic, methylmalonic, and isovaleric acidemias could be grouped under a "ketoacidotic/acidemic/acid blood" category. These categories are examples and alternative assemblages or designations can be created to maximize their political impact. Grouping rare disorders with unpronounceable names into categories with comprehensible names can accelerate policymakers' understanding and implementation of MS/MS technology for newborn screening programs.


Supplemental Neonatal Screening from Acylcarnitine Analysis Using Tandem Mass Spectrometry: Reliability and Specificity Charles R. Roe, M.D.; Baylor University Medical Center, Dallas, Texas

The majority of the disorders detectable by tandem mass spectrometry (MS/MS) are characterized by multiple clinical phenotypes.****** This phenotypic complexity is compounded by acylcarnitine profiles, which are not always specific for a single disease. Diseases that are associated with identical or overlapping acylcarnitine profiles include the following examples:

  • Long-chain hydroxy acylcarnitine deficiency is indistinguishable from trifunctional protein deficiency.
  • Carnitine palmitoyltransferase II is indistinguishable from carnitine acylcarnitine translocase deficiencies.
  • The observation of an increase in 3-hydroxy-isovaleryl-carnitine raises the possibilities of 3-methylcrotonyl-CoA carboxylase, 3-methylglutaconic, hydro-xymethylglutaryl-CoA lyase, and multiple carboxylase deficiencies.
  • An increase in an acylcarnitine containing five carbons can indicate either isovaleric acidemia or the recently identified S-2-methylbutyryl-CoA dehydrogenase deficiency.

Only three disorders exist for which the acylcarnitine profile is completely disease-specific: medium-chain acyl-CoA dehydrogenase deficiency, glutaric aciduria type I, and malonic aciduria. Distinctions among diseases that have similar acylcarnitine profiles can be aided by knowledge of the patient's clinical course, but that information might not be available for interpreting abnormal test results before symptom onset. Recent descriptions of inherited disorders that have the same blood-spot acylcarnitine profile emphasize the need for additional documentation to accurately diagnose the specific disorder and subsequently manage treatment appropriately. Similarly, in vitro demonstrations have been reported of distinct acylcarnitine profiles for different clinical phenotypes of the same disease, even with the same mutation.§ Acylcarnitine profiles from newborn blood spots do not correlate with these clinical phenotypes. Finally, not all diseases that are potentially detectable by MS/MS acylcarnitine analysis have been observed among newborns.

Tandem Mass Spectrometry in the New South Wales Newborn Screening Program Bridget Wilcken, M.B., Ch.B.; Veronica Wiley, Ph.D.; New Children's Hospital, Sydney, Australia

We introduced electrospray tandem mass spectrometry (MS/MS) into the New South Wales newborn screening program in 1998.******* We have screened 196,000 babies, usually on day 3 of life. A total of 50 babies had confirmed defects, approximately the expected rate based on 20 years' previous experience in our statewide biochemical genetics laboratory or on known mutation frequencies: phenylketonuria 28 (21 expected); biopterin defects, two; other aminoacidopathies, five; organic aciduria disorder, three; medium-chain acyl-CoA dehydrogenase deficiency, six (six expected); other fatty acid oxidation disorders, two; noninborn errors (neonatal hepatitis or maternal B12 deficiency), four. Three were known false-negatives, cases of nonketotic hyerglycinaemia, tyrosinaemia type I, and cobalamin C defect. Only 210 repeat samples were requested from babies not having a proven defect. The substantial number of primary analytes used gives a risk for an unacceptable recall rate. Analysis of many retrospective newborn samples from cases later diagnosed clinically is required. An experienced biochemical genetics laboratory and quality-assurance programs are requirements for diagnosis and later monitoring. Where the screening laboratory is not linked to a biochemical genetics laboratory, record keeping will be more difficult because final diagnosis takes time, and the risk exists of recording cases that are really false-positives. Because diagnosis of clinically nonsignificant conditions can be a problem where experienced teams are not available, a limited number of specified disorders should be sought in the first instance. Treatment of asymptomatic babies has not proven to be a problem and is not different from the familiar phenylketonuria situation. Certain potentially detectable disorders are extremely rare. Follow-up should be done only by a clinical biochemical geneticist experienced in managing metabolic disorders, with multidisciplinary care, expert dietician, and biochemical genetics laboratory support. Evaluation of cost-benefit and reduction in morbidity and mortality will be extremely difficult unless accurate, comprehensive records are available for the state or area. We have records of all relevant inborn errors of metabolism detected in New South Wales over the last 20 years. We will use historical controls to come to an estimate of benefit. Randomized controlled trials for the most part will not be possible because of the rarity of individual conditions.

University of Wisconsin Biochemical Genetics Program Jon Wolff, M.D.; Sandra van Calcar, M.S.; Kristine K. Hanson, M.S.; University of Wisconsin, Madison, Wisconsin

In June 1999, the University of Wisconsin Biochemical Genetics Program became involved in the follow-up of abnormal acylcarnitine test results. Our team includes a board-certified biochemical geneticist, a nutritionist, and a genetics counselor. The state laboratory reports all abnormal test results to us and the physician of record. Immediately, our geneticist contacts the child's physician to discuss the diagnosis and arrange follow-up testing. For all disorders, follow-up testing includes urinary organic acids and repeat acylcarnitines, which usually takes 12 weeks. During this time, the physician is asked to watch for signs of metabolic crisis and instructed how to treat a patient having an acute episode (i.e., with intravenous fluids or glucose). The genetic counselor identifies or creates resource materials for the physician and family and is available to the family during this time. If an abnormal test result is confirmed by follow-up testing, the child is examined and the parents provided nutrition consultation and genetic counseling. Our patient treatment for confirmed short- and medium-chain acyl-CoA dehydrogenase disorders includes four components,

  • avoidance of fasting;
  • administration of carnitine at 50 mg/kg/day, divided, 3 times/day;
  • consumption of a moderately low-fat diet (e.g., 30% kcal from fat); and
  • prompt treatment for vomiting episodes with intravenous fluids or glucose.

An emergency protocol is written for each child and given to parents for delivery to emergency department personnel, if needed. Because increasing physician awareness and creating resource materials for physicians and patients' families is a high priority, we are developing an education module for physicians.

* Source: Rashed MS, Bucknall MP, Little D, et al. Screening blood spots for inborn errors of metabolism by electrospray tandem mass spectrometry with a microplate batch process and a computer algorithm for automated flagging of abnormal profiles. Clin Chem 1997;43:112941.

** Source: Hubbard AR, Margetts SML, Barrowcliffe TW. International normalized ratio determination using calibrated reference plasmas. Br J Haematol 1997;98:748.

*** Punching is a procedure that takes an aliquot portion (e.g., 1/8 inch) from the dried blood spot contained on the filter paper.

**** Source: Institute of Medicine. Future of public health. Washington, DC: National Academy Press, 1988.

Source: Gordis L. Using epidemiology to evaluate health services. In: Gordis L. Epidemiology. 2nd ed. Philadelphia: WB Saunders Co., 1996:21728.

***** Source: Institute of Medicine/Committee for the Study of the Future of Public Health. Promoting safe and effective genetic testing in the United States: final report of the Task Force on Genetic Testing. Holtzman NA, Watson MS, eds. Washington, DC: National Academy Press, 1998:1180.

Source: Wang SS, Fernhoff PM, Hannon WH, Khoury MJ. Medium chain acyl Co-A dehydrogenase deficiency human genome epidemiology review. Genetics in Medicine 1999;1:3329.

******Source: Roe CR, Ding JH. Disorders of mitochondrial function. In: Scriver CR, Beaudet AL, Sly WS, eds. Metabolic and molecular bases of inherited diseases. New York: McGraw-Hill. 2001.

†† Sources: Roe CR, Cederbaum SD, Roe DS, Mardach R, Galindo A, Sweetman L. Isolated isobutyryl-CoA dehydrogenase deficiency: an unrecognized defect in human valine metabolism. Mol Genet Metab 1998;65:26471; and Gibson KM, Burlingame TG, Hogema B, et al. 2-methylbutyryl-coenzyme A dehydrogenase deficiency: a new inborn error of L-isoleucine metabolism. Pediatr Res 2000;47:8303.

§ Sources: Roe CR, Roe DS. Recent developments in the investigation of inherited metabolic disorders using cultured human cells. Mol Genet Metab 1999;68:24357; and Roe CR, Roe DS. Detection of gene defects in branched-chain amino acid metabolism by tandem mass spectrometry of carnitine esters produced by cultured fibroblasts. In: Harris RA, Sokatch JR, eds. Methods in enzymology. Vol 324. San Diego, California: Academic Press, 2000:42431.

*******Source: Wiley V, Carpenter K, Wilcken B. Newborn screening with tandem mass-spectrometry: 12 months' experience in NSW Australia. Acta Paediatr Suppl 1999:432:4851.

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Morbidity and Mortality Weekly Report
Centers for Disease Control and Prevention
1600 Clifton Rd, MailStop E-90, Atlanta, GA 30333, U.S.A


Department of Health
and Human Services

This page last reviewed 5/2/01