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Articles |
1
Neo Gen Screening, 110 Roessler Rd., Suite 200 D, Pittsburgh, PA 15220.
2
Mass Spectrometry Facility, Division of Medical
Genetics, Department of Pediatrics, Duke University Medical Center, Box
14991, Research Triangle Park, NC 27709.
a Author for correspondence. Fax 412-341-8926; e-mail dhchace{at}Neogenscreening.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The increase of medium chain acyl-CoA esters resulting from the enzymatic block leads to increased medium-chain fatty acids, dicarboxylic acids, acylglycines, and medium-chain acylcarnitines in plasma and urine. Strategies proposed for the identification of MCAD deficiency for newborn screening have included molecular screening for the common A985G mutation (7) or recognition of metabolites such as diagnostic increases of cis-4-decenoic acid (8), hexanoylglycine (9), or specific medium-chain acylcarnitines in blood spots (10). In a diagnostic clinical setting, analysis of acylcarnitines by tandem mass spectrometry (MS-MS) as their methyl esters allowed the diagnostic recognition of all patients with MCAD deficiency regardless of the underlying mutation, symptomatic state, or treatment (11). Patients with MCAD deficiency have increased octanoylcarnitine (>0.3 µmol/L) and an increased octanoylcarnitine-to-decanoylcarnitine ratio (>5). Similar results were found in the analysis of newborn blood cards.
Analysis of acylcarnitines after derivatization as butyl esters was recently proposed (12). Derivatization as butyl esters has the advantage to also allow amino acid analysis on the same sample preparation. The analysis of amino acids as their butyl esters has been validated for newborn screening of phenylketonuria, tyrosinemia, maple syrup urine disease, and homocystinuria (12)(13)(14)(15). In this study we validate the screening methodology for the analysis of acylcarnitines as their butyl esters from dried blood spots for the diagnosis of MCAD deficiency in newborns. Quantitative data obtained from prospective newborn screening are compared with data from newborns with MCAD deficiency. The evolution of the diagnostic acylcarnitines over the first days of life is investigated and compared with those in affected children. Finally, we report the first results of real-time newborn screening using this method in Pennsylvania and North Carolina.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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blood specimen collection
The specimens tested consisted of: (a) 16 500 dried
blood spots from the North Carolina Division of Laboratory Services
Newborn Screening Program obtained primarily from the Research Triangle
region between 1993 and 1995; (b) a collection of blood
spots from healthy newborns and from newborns confirmed with MCAD
deficiency; these collections are part of a larger prospectively
screened group of 267 303 samples from the supplemental newborn
screening program at Neo Gen Screening in Pennsylvania that covered
primarily Western and Central Pennsylvania between September 1992 and
January 1997; and (c) a collection of blood spots of
patients found to have MCAD deficiency from the medical genetics
laboratory at the Mass Spectrometry Facility, Duke University Medical
Center. All MCAD specimens were retrieved from storage of <3 years.
All specimens from the North Carolina Newborn Screening Program, Neo
Gen Screening, and all the specimens received in the medical genetics
laboratory were collected or prepared on S&S Grade 903 filter paper
(Schleicher and Schuell).
sample preparation
The semiautomated preparation of butyl ester derivatives of
acylcarnitines and amino acids from blood spots consisted of a simple
solvent extraction and derivatization procedure that takes ~2.5 h for
60 samples. This method has been described previously
(13)(14)(15) with the following addition. The methanol stock
solution that contained amino acid internal calibrators as described
previously also contained the following additional internal
calibrators: [2H3]acetylcarnitine
(5 µmol/L), [2H3]propion-ylcarnitine (1
µmol/L), [2H3]octanoylcarnitine (1
µmol/L), and [2H3]palmitoylcarnitine (2
µmol/L). In brief, two 3/16-in. diameter punches
from blood spots equivalent to 15.2 µL of whole blood
(16) were extracted with 400 µL of this stock solution
and evaporated to dryness at 50 °C under a gentle stream of
nitrogen. Fifty microliters of butanolic HCl (3 mol/L) was added to
each sample; the samples were then incubated at 65 °C for 15 min.
After evaporation of excess butanolic HCl (3 mol/L), the derivatized
samples were reconstituted with 35 µL of 1:1 (by vol)
methanol:glycerol with 1 g/L sodium octyl sulfate for analysis by
MS-MS.
method validation
To estimate the linearity of this assay, four separate aliquots of
whole blood from single donors were enriched with either acetyl-,
hexanoyl-, octanoyl-, or decanoylcarnitine. In addition to unenriched
whole blood, the following enrichments were prepared (five-point
calibration curve): acetylcarnitine, 2.5–50 µmol/L;
hexanoylcarnitine, 0.5–10 µmol/L, octanoylcarnitine, 0.5–10
µmol/L; decanoylcarnitine, 0.5–10 µmol/L. These samples were then
spotted on filter paper and dried overnight. For recovery studies
(extraction efficiency) of acylcarnitines from blood spots, aliquots of
whole blood were prepared from a single donor pool and enriched with 0,
2.5, 10, and 80 µmol/L of acetylcarnitine; 0, 0.5, 2, and 8 µmol/L
of octanoylcarnitine; and 0, 2, 10, and 20 µmol/L each of
hexanoylcarnitine and decanoylcarnitine. Before spotting, each set of
aliquots was divided into two groups. One group of samples was spotted
on filter paper, dried overnight, and prepared as described
above. To the second group of whole-blood samples,
[2H3]acetylcarnitine and
[2H3]octanoylcarnitine were added to each
aliquot (sample set) at the concentrations described above and mixed
well. These whole-blood samples, containing internal calibrators, were
spotted on filter paper, dried overnight, and prepared by extraction
with pure methanol containing no internal calibrators. The remainder of
the sample preparation procedure was followed as described above.
Instrument variability was measured by performing 10 replicate injections of one sample. Assay variability was measured by performing analyses of 10 control samples prepared from the same blood on one day (intraday variability) and over several weeks (interday variability).
mass spectrometry
A VG Quattro quadrupole tandem mass spectrometer with a
laboratory-based data system (Micromass) was used and operated in the
static liquid secondary ionization mode. This mode incorporates an ion
source containing a cesium ion gun operating at 10 keV and a manually
operated insertion probe. Positively charged molecules are detected
after separation in the first mass analyzer region (MS1) and in the
final mass analyzer region (MS2). An intermediate hexapole located
between the first quadrupole and the second quadrupole is used as the
collision region into which argon gas is introduced. Tuning of the
instrument was optimized with a solution containing deuterium-labeled
calibrators prepared as butyl esters as described previously
(12).
Product ion scans were produced by focusing MS1 on the molecular masses (M+H)+ of the butyl ester of octanoylcarnitine (m/z 344) and its internal calibrator [2H3]octanoylcarnitine (m/z 347), while MS2 was used to scan fragment ions between m/z 50 and m/z 400. Mass spectra showing the fragmentation of octanoylcarnitine and its internal calibrator [2H3]octanoylcarnitine were obtained. Parent ion scans of 85 Da were produced by scanning MS1 from m/z 255–550 while focusing MS2 on a single common product ion at m/z 85 produced by all acylcarnitine butyl esters. This resulted in a spectrum of parent ions (molecular ions) corresponding to (M + H)+.
Quantification of octanoylcarnitine was achieved by converting the ion abundance ratios of octanoylcarnitine to [2H3]octanoylcarnitine (m/z 344:347) and then interpolating the concentration values for octanoylcarnitine by reference to a calibration curve. A calibration curve was generated from the analyses of blood spots containing serially added fixed concentrations of octanoylcarnitine. Quantification of acetylcarnitine (m/z 260) was achieved similarly with [2H3]acetylcarnitine as the internal calibrator (m/z 260:263). No isotopically labeled internal calibrator was available for hexanoylcarnitine and decanoylcarnitine. Quantification of these compounds was achieved by calculating the ion abundance ratios of the pure unlabeled compound relative to [2H3]octanoylcarnitine (i.e., hexanoylcarnitine, m/z 316:347, and decanoylcarnitine, m/z 372:347). A calibration curve was generated from the analyses of blood spots containing serially added fixed concentrations of either of these calibrators (hexanoyl- and decanoylcarnitine). No pure material was available for decenoylcarnitine. An approximation of the concentration of decenoylcarnitine was achieved by extrapolation from the calibration curve of the ion abundance ratios of decanoylcarnitine to [2H3]octanoylcarnitine.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. All other butyl esters of acylcarnitines (such as
acetylcarnitine, hexanoylcarnitine, and decanoylcarnitine) share a
similar fragmentation pattern with a common fragment ion at 85 Da. The
fragmentation process, which is shown schematically in Fig. 2
, involves loss of the fatty acid, trimethylamine, and butene,
resulting in the generation of a stable fragment ion with
m/z 85. Other acylcarnitine (acetyl-, hexanoyl-, and
decanoylcarnitine) butyl esters exhibited similar fragmentation,
resulting in a prominent fragment ion at m/z 85. Therefore,
to analyze all acylcarnitines, the tandem mass spectrometer is set up
such that MS1 will scan the entire mass range (m/z 225–550)
while MS2 is set up to monitor m/z 85, the mass of the
fragment ion common to acylcarnitine butyl esters. Product ions of 85
Da from acylcarnitine butyl esters are detected in MS2. Note that the
mass of the parent ion is shown on the mass spectra although the
product ion is actually detected. This method of MS-MS data acquisition
is known as precursor ion scanning.
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In biological samples, the parent ion scan detects short-chain
acylcarnitines such as propionylcarnitine and butyrylcarnitine,
long-chain acylcarnitines such as palmitoylcarnitine and
linoleylcarnitine, dicarboxylic acid acylcarnitines such as
glutarylcarnitine, or hydroxyacylcarnitines such as
3-hydroxyisovalerylcarnitine (12). Fig. 1
shows the parent
ion spectrum of the molecular ion (precursor ion) for pure
octanoylcarnitine and [2H3]octanoylcarnitine.
The parent ion spectrum of octanoylcarnitine and
[2H3]octanoylcarnitine are not single peaks.
In addition to the dominant molecular ion, there are small peaks at
m/z 288 and m/z 291 that may be the result of
incomplete derivatization of acylcarnitines or of secondary
fragmentation.
Figure
3 (top) shows a representative acylcarnitine profile of a blood
spot from a fresh, normal newborn screening filter paper card obtained
by using the parents of 85 Da scan function. Ion signals at
representative masses of several acylcarnitines are m/z 260
(acetylcarnitine), m/z 274 (propionylcarnitine),
m/z 456 (palmitoylcarnitine), and m/z 482
(linoleylcarnitine), and the internal calibrators at m/z 263
([2H3]acetylcarnitine),
m/z 277 ([2H3]propionylcarnitine),
m/z 347 ([2H3]octanoylcarnitine),
and m/z 459
([2H3]palmitoylcarnitine). The only
significant signals detected are from acylcarnitines and
their added internal calibrators. Octanoylcarnitine is generally
present at very low concentrations in healthy newborn blood samples and
the signal representative of this component is often at the limits of
detection. Fig. 3
(middle) shows an acylcarnitine profile from a
newborn screening blood card from an MCAD patient subsequently
confirmed by DNA analysis as a homozygote for the common A985G
mutation. The ion signal of octanoylcarnitine (m/z 344) is
clearly increased higher than the internal calibrator ion signal at
m/z 347. There is also an increase in the signal intensities
of hexanoylcarnitine (m/z 316), decenoylcarnitine
(m/z 370), and decanoylcarnitine (m/z 372). Fig. 3
(bottom) shows an acylcarnitine profile of a blood spot from a
newborn screening filter paper card of an MCAD patient subsequently
confirmed by DNA analysis to be a compound heterozygote with one copy
of the gene for the A985G mutation present. Similar increases of
diagnostically important acylcarnitines are seen in Fig. 3
(middle and
bottom panels).
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assay calibration and limits of detection
Calibration curves for acetylcarnitine and octanoylcarnitine were
generated with standard isotope dilution techniques. The ion
signals corresponding to acetyl- or octanoylcarnitine and their
respective deuterium-labeled internal calibrators were plotted as a
function of the concentration of added acetyl- or octanoylcarnitine to
blood. The calibration curves for acetyl- and octanoylcarnitine were
linear over the concentration ranges of 0–50 and 0–10 µmol/L,
respectively. The regression analyses and statistical data are
presented in Table 1
. Detection limits were based on a signal-to-noise ratio of 3:1.
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Calibration curves for hexanoyl- and decanoylcarnitine were generated
with slight variations of standard isotope dilution techniques
whereby the internal calibrator is a closely related isotope of the
calibrator. The ion signal ratios of hexanoyl- or decanoylcarnitine to
the internal calibrator,
[2H3]octanoylcarnitine, were plotted as a
function of the concentration of added hexanoyl- or decanoylcarnitine
to blood. The calibration curves for hexanoylcarnitine and
decanoylcarnitine were each linear over the concentration range of
0–10 µmol/L (Table 1
). These results indicate excellent linearity of
the assay within these ranges seen in most normal and all pathologic
samples.
analytical recovery, precision, and accuracy
The analytical recoveries of acetyl-, hexanoyl-, octanoyl-, and
decanoylcarnitine added to blood were determined in triplicate. These
results show good recovery (Table 2
). The analytical imprecision, CV, determined by 10 replicate
analyses of the derivatized product of a single sample for acetyl-,
hexanoyl-, octanoyl-, and decanoylcarnitine is described in Table 3
. Overall precision of the assay calculated by replicate
analyses was <8% of the same normal blood sample on the same day and
on different days. The intraday and interday precision (CV) for
acetyl-, hexanoyl-, octanoyl-, and decanoylcarnitine and the ratios of
octanoylcarnitine to acetylcarnitine, hexanoylcarnitine to
acetylcarnitine, and decanoylcarnitine to acetylcarnitine was <15%
for all analytes. This is sufficient for the correct differentiation
between normal and pathologic samples. The similar intra- and interday
imprecision illustrates the stability of the method.
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analysis of blood specimen collections
The range of normal values was determined in a sampling of 113 blood
spots from the group of normal neonatal blood spots from Duke
University Medical Center (Table 4
). The concentration of acetylcarnitine, the most prominent
physiologic metabolite, was 22.3 mmol/L ± 13.5 (mean ± SD).
The minimum concentration observed was 3.5 mmol/L, and the maximum
concentration 79.5 mmol/L. In these normal blood spots, the
concentrations of the other pathologic acylcarnitines (hexanoyl-,
octanoyl-, decanoyl-, and decenoylcarnitine) were very low, the median
always being below the detection limit. For diagnostic distinction from
the increased concentrations observed in blood spots from patients with
MCAD deficiency, the upper range of normal must be determined. Table 4
describes the number of samples above the detection limit, and
describes the upper ranges observed in normal blood spots as 95th
percentile and maximum concentration observed. For hexanoyl-,
octanoyl-, and decanoylcarnitine the upper quarter of the normal
concentration range was within the sensitivity of the assay, and upper
limits as quantified were well below 0.3 µmol/L. In no normal blood
spot was decenoylcarnitine detected.
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Sixteen original newborn blood spots collected <72 h after birth from
neonates with MCAD deficiency verified by DNA mutation analysis were
retrieved from both Neo Gen Screening and Duke University Medical
Center. Ten patients were homozygous for the A985G mutation and six
were compound heterozygotes. In these patient samples, all metabolites
were well within the dynamic range of the assay, as shown in Table 4
.
All pathologic acylcarnitines were above the upper values of normal
controls, allowing easy diagnostic distinction.
To evaluate the influence of patient age on the concentration of the
acylcarnitines in MCAD deficiency, we analyzed blood spots from 16
older patients outside the neonatal age. Ages ranged from 8 days to 11
years, and eight of these patients were homozygous for the A985G
mutation. In these patients, the diagnostic acylcarnitines (hexanoyl-,
octanoyl-, decenoyl-, and decanoylcarnitine) were increased but to a
lesser degree than observed in the neonatal period (P
<0.0001, Mann–Whitney U-test) (Table 4
). Octanoylcarnitine
was still >0.3 µmol/L but the diagnostic distinction more difficult.
These samples were analyzed after storage in this laboratory and were
not the results of the original analysis. Acetylcarnitine may degrade
up to 50%, propionylcarnitine degrades <50%, and palmitoylcarnitine
<10% over 3 years (17).
Previous studies have demonstrated the diagnostic advantage of the
ratio of metabolites in the same sample. In MCAD deficiency, previous
studies have related the concentration of octanoylcarnitine to
acetylcarnitine (11)(18) or to
decanoylcarnitine (11). Table 5
shows the ratios of the molar concentrations of the diagnostic
metabolites to acetylcarnitine, and the molar ratio of octanoyl- to
decanoylcarnitine. Both the ratios of octanoyl- to acetylcarnitine
(C8/C2) and of octanoyl- to decanoylcarnitine (C8/C10) were clearly
increased in all patients with MCAD deficiency, regardless of age, when
compared with the neonatal control values (Table 5
). This distinction
was not so pronounced for the metabolite ratios C6/C2 and C10/C2.
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Prospective newborn screening of 283 803 infants by Neo Gen Screening in Pennsylvania and Duke University Medical Center in North Carolina showed a disease frequency for MCAD of 1:17 706. Of these, nine of 16 MCAD patients were homozygous for the A985G mutation, the remaining being compound heterozygotes with one copy of this common mutation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The application of the stable isotope dilution techniques to the analysis of acylcarnitines as their butyl esters from newborn blood spots proved a highly accurate technique, with complete extraction from the filter paper, and an imprecision of <10%. The assay is robust, with intraday and interday imprecision of <15%. Interference due to overlapping components such as a small contribution of glutamate to the m/z 260 peak has a generally negligible effect on the accuracy of the quantification for its relevance to diagnostic application. Long-term sample storage leads primarily to the decline in the concentration of short-chain acylcarnitine due to sample degradation, but this is not relevant to the practice of newborn screening (17).
For accurate diagnosis the working range of an assay must cover the diagnostic region. The concentration of the pathologic metabolites hexanoyl-, octanoyl-, decenoyl-, and decanoylcarnitine in samples from healthy controls is very low, and is below the detection limit in approximately two-thirds of the population. However, the assay accurately detects and quantifies the upper third of the concentration range of the healthy population, and the whole range of concentrations found in patients with MCAD deficiency. This allows the development of diagnostic criteria, and the evaluation of their accuracy. In patients with MCAD deficiency all four metabolites are generally increased, with octanoylcarnitine always being well above the upper range of that seen in healthy controls. The concentrations of the pathologic metabolites are higher in the newborn period than those observed in older patients in a routine diagnostic laboratory setting. This difference is more pronounced than can be explained by the degradation due to sample storage of the retrospectively analyzed samples of older MCAD patients. The progressive development of carnitine deficiency through renal loss represents one possible explanation for the lower values observed in older patients. These higher metabolite concentrations in the newborn period facilitate the detection of MCAD deficiency. Octanoylcarnitine concentrations >0.3 µmol/L and a C8/C10 ratio >2 in patients of all age groups have to be considered as strongly indicative of MCAD deficiency, and is aided by C8/C2 >0.1. These criteria can easily be incorporated in a computer program for assistance in recognizing those patients who require further evaluation for a possible diagnosis of MCAD deficiency.
The higher concentrations of acylcarnitines in the neonatal period allow a higher cutoff. This can be advantageous since small increases can occur in normal heterozygote carriers, particularly during the neonatal period when diagnostic metabolites are more pronounced. Of the 267 303 samples screened at Neo Gen Screening, five samples that had small increases in octanoylcarnitine that were less than the diagnostic cutoff of 0.3 µmol/L were found to have a single copy of A985G mutation for MCAD. No increase in octanoylcarnitine was found upon analysis of a second sample. So far no additional mutations have been recognized. Further study on the octanoylcarnitine concentration of obligate heterozygotes is under investigation. However, using the diagnostic criteria of 0.3 µmol/L for octanoylcarnitine, the false-positive rate for MCAD deficiency is <0.01%.
Application of this technology to prospective newborn screening in a set of 283 803 samples from Pennsylvania and North Carolina revealed a disease frequency of 1:17 706. As previously reported [20], this incidence is considerably higher than estimates based on the gene frequency of the A985G mutation (5). This is not attributable primarily to high-risk subpopulations such as the Amish and Mennonites, although MCAD has been reported in these populations in Pennsylvania. This high frequency is also observed in the North Carolina samples. Prospective studies will have to establish to what extent these neonatally identified patients will become similarly symptomatic. If screening was DNA-based, the high heterozygote frequency of A985G mutation (1:84 Caucasians in North Carolina) would require frequent retesting by a second technique such as the analysis of acylcarnitines.
Some patients with MCAD deficiency (up to 7%) present acutely in the neonatal period (2)(21)(22). These patients have typically presented on the third or the fourth day of life. Rapid diagnostic recognition will be required to timely ascertain these patients. The high concentration of the diagnostic acylcarnitines in the neonatal blood spots of affected patients indicates that early diagnostic recognition will be feasible, provided efficient sample collection and processing is available.
Because of the high incidence, the significant risk for death or serious sequelae, and the ease and efficacy of treatment, this disorder is considered a good candidate for newborn screening (6). Real-time newborn screening for MCAD deficiency by analysis of acylcarnitines is now technically feasible. Prospective studies will have to establish how efficacious these screening programs will be in the ultimate prevention of mortality and morbidity. This obviously will require appropriate follow-up and treatment in addition to accurate and timely recognition.
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Footnotes |
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The following articles in journals at HighWire Press have cited this article:
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  S. E Reuter, A. M Evans, D. H Chace, and G. Fornasini Determination of the reference range of endogenous plasma carnitines in healthy adults Ann Clin Biochem, November 1, 2008; 45(6): 585 - 592. [Abstract] [Full Text] [PDF]
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 H.-W. Hsu, T. H. Zytkovicz, A. M. Comeau, A. W. Strauss, D. Marsden, V. E. Shih, G. F. Grady, and R. B. Eaton Spectrum of Medium-Chain Acyl-CoA Dehydrogenase Deficiency Detected by Newborn Screening Pediatrics, May 1, 2008; 121(5): e1108 - e1114. [Abstract] [Full Text] [PDF]
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 S. Ho, Z. Lukacs, G. F. Hoffmann, M. Lindner, and T. Wetter Feature Construction Can Improve Diagnostic Criteria for High-Dimensional Metabolic Data in Newborn Screening for Medium-Chain Acyl-CoA Dehydrogenase Deficiency Clin. Chem., July 1, 2007; 53(7): 1330 - 1337. [Abstract] [Full Text] [PDF]
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K. A. Strnadova, M. Holub, A. Muhl, G. Heinze, R. Ratschmann, H. Mascher, S. Stockler-Ipsiroglu, F. Waldhauser, F. Votava, J. Lebl, et al. Long-Term Stability of Amino Acids and Acylcarnitines in Dried Blood Spots Clin. Chem., April 1, 2007; 53(4): 717 - 722. [Abstract] [Full Text] [PDF]
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C. I. Kaye and and the Committee on Genetics Newborn Screening Fact Sheets Pediatrics, September 1, 2006; 118(3): e934 - e963. [Abstract] [Full Text] [PDF]
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B. A. Tarini, D. A. Christakis, and H. G. Welch State Newborn Screening in the Tandem Mass Spectrometry Era: More Tests, More False-Positive Results Pediatrics, August 1, 2006; 118(2): 448 - 456. [Abstract] [Full Text] [PDF]
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D. S. Millington Newborn Screening for Lysosomal Storage Disorders Clin. Chem., May 1, 2005; 51(5): 808 - 809. [Full Text] [PDF]
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C. T. Cavedon, P. Bourdoux, K. Mertens, H. V. Van Thi, N. Herremans, C. de Laet, and P. Goyens Age-Related Variations in Acylcarnitine and Free Carnitine Concentrations Measured by Tandem Mass Spectrometry Clin. Chem., April 1, 2005; 51(4): 745 - 752. [Abstract] [Full Text] [PDF]
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R. Rej Clinical Chemistry through Clinical Chemistry: A Journal Timeline Clin. Chem., December 1, 2004; 50(12): 2415 - 2458. [Abstract] [Full Text] [PDF]
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D. H. Chace, T. A. Kalas, and E. W. Naylor Use of Tandem Mass Spectrometry for Multianalyte Screening of Dried Blood Specimens from Newborns Clin. Chem., November 1, 2003; 49(11): 1797 - 1817. [Abstract] [Full Text] [PDF]
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L. N. Venditti, C. P. Venditti, G. T. Berry, P. B. Kaplan, E. M. Kaye, H. Glick, and C. A. Stanley Newborn Screening by Tandem Mass Spectrometry for Medium-Chain Acyl-CoA Dehydrogenase Deficiency: A Cost-Effectiveness Analysis Pediatrics, November 1, 2003; 112(5): 1005 - 1015. [Abstract] [Full Text] [PDF]
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A. Schulze, M. Lindner, D. Kohlmuller, K. Olgemoller, E. Mayatepek, and G. F. Hoffmann Expanded Newborn Screening for Inborn Errors of Metabolism by Electrospray Ionization-Tandem Mass Spectrometry: Results, Outcome, and Implications Pediatrics, June 1, 2003; 111(6): 1399 - 1406. [Abstract] [Full Text] [PDF]
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 M. J. Khoury, L. L. McCabe, and E. R.B. McCabe Population Screening in the Age of Genomic Medicine N. Engl. J. Med., January 2, 2003; 348(1): 50 - 58. [Full Text] [PDF]
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L. Sweetman Newborn Screening by Tandem Mass Spectrometry: Gaining Experience Clin. Chem., November 1, 2001; 47(11): 1937 - 1938. [Full Text] [PDF]
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T. H. Zytkovicz, E. F. Fitzgerald, D. Marsden, C. A. Larson, V. E. Shih, D. M. Johnson, A. W. Strauss, A. M. Comeau, R. B. Eaton, and G. F. Grady Tandem Mass Spectrometric Analysis for Amino, Organic, and Fatty Acid Disorders in Newborn Dried Blood Spots: A Two-Year Summary from the New England Newborn Screening Program Clin. Chem., November 1, 2001; 47(11): 1945 - 1955. [Abstract] [Full Text] [PDF]
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 G. M. Enns Newborn Screening by Tandem Mass Spectrometry NeoReviews, August 1, 2001; 2(8): e201 - 207. [Full Text] [PDF]
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D. H. Chace, J. C. DiPerna, B. L. Mitchell, B. Sgroi, L. F. Hofman, and E. W. Naylor Electrospray Tandem Mass Spectrometry for Analysis of Acylcarnitines in Dried Postmortem Blood Specimens Collected at Autopsy from Infants with Unexplained Cause of Death Clin. Chem., July 1, 2001; 47(7): 1166 - 1182. [Abstract] [Full Text] [PDF]
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P. M. Jones, M. Moffitt, D. Joseph, P. A. Harthcock, R. L. Boriack, J. A. Ibdah, A. W. Strauss, and M. J. Bennett Accumulation of Free 3-Hydroxy Fatty Acids in the Culture Media of Fibroblasts from Patients Deficient in Long-Chain L-3-Hydroxyacyl-CoA Dehydrogenase: A Useful Diagnostic Aid Clin. Chem., July 1, 2001; 47(7): 1190 - 1194. [Abstract] [Full Text] [PDF]
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J. C. Wood, M. J. Magera, P. Rinaldo, M. R. Seashore, A. W. Strauss, and A. Friedman Diagnosis of Very Long Chain Acyl-Dehydrogenase Deficiency From an Infant's Newborn Screening Card Pediatrics, July 1, 2001; 108(1): e19 - 19. [Abstract] [Full Text] [PDF]
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 D. Matern and M. J. Magera Mass Spectrometry Methods for Metabolic and Health Assessment J. Nutr., May 1, 2001; 131(5): 1615S - 1620. [Abstract] [Full Text]
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D. H. Chace, J. C. DiPerna, B. W. Adam, and W. H. Hannon Errors Caused by the Use of D,L-Octanoylcarnitine for Blood-Spot Calibrators Clin. Chem., April 1, 2001; 47(4): 758 - 760. [Full Text] [PDF]
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S. Albers and H. L. Levy One More Thought on Sudden Infant Death Syndrome Pediatrics, April 1, 2001; 107(4): 809 - 809. [Full Text]
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M. J. Magera, J. K. Helgeson, D. Matern, and P. Rinaldo Methylmalonic Acid Measured in Plasma and Urine by Stable-Isotope Dilution and Electrospray Tandem Mass Spectrometry Clin. Chem., November 1, 2000; 46(11): 1804 - 1810. [Abstract] [Full Text] [PDF]
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