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Articles |
1
Departments of Biological and Medical Research and
2
Pediatrics, King Faisal Specialist Hospital and Research Centre, PO Box 3354, Riyadh 11211, Saudi Arabia.
a Author for correspondence. Fax 966-1-442-7858; e-mail rashed{at}kfshrc.edu.sa
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: amino acids • organic acidemia • defects of fatty acid oxidation • inherited disorders • neonatal screening
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For >30 years in developed countries, the Guthrie qualitative bacterial inhibition test for phenylalanine in blood spots has formed the basis for successful national neonatal screening programs for phenylketonuria (PKU) (12). Similar methods have been adopted on a limited scale for diseases such as maple syrup urine disease, isolated hypermethioninemia, homocystinuria, and galactosemia (10)(13). The more comprehensive quantitative determination of plasma amino acids by HPLC is usually reserved for selective screening of high-risk infants, for confirmation of neonatal screening results, for follow-up of treatment, and for identification of other aminoacidopathies (14)(15).
Organic acidemias caused by mitochondrial enzyme defects in the catabolism of branched-chain amino acids and fatty acid oxidation defects constitute a group of >20 disorders in which acyl-CoA esters accumulate in the mitochondria. In these disorders, carnitine plays a key role, removing the potentially toxic acyl-CoA esters through the formation of acylcarnitine esters and thereby releasing coenzyme A and restoring mitochondrial homeostasis (16)(17). This results in increased concentrations of circulating acylcarnitines, increased excretion of acylcarnitines in urine, and secondary carnitine deficiency (18). Therefore, metabolic profiling of free carnitine and acylcarnitine in plasma, blood spots, or urine by various spectrophotometric, radiochemical, or mass-spectrometric methods provides a powerful selective screening tool for these disorders (19)(20)(21)(22)(23)(24)(25).
However, the above methods for either neonatal or selective screening of metabolic disorders share some, if not all, of the following drawbacks: (a) narrow spectrum of diseases covered by each method; (b) high false-positive rates; (c) low specificity or sensitivity (or both); (d) laborious sample preparation; and (e) long analytical time. Millington et al. put forward the concept of broad-spectrum screening for IEMs by fast atom bombardment tandem mass spectrometry (FAB-MS/MS) for metabolic profiling of acylcarnitines from plasma or dried blood spots (26)(27)(28). MS/MS, by eliminating the need for chromatographic separation, shortens analysis time to <2 min, thereby offering the possibility of high sample throughput. The specificity and the accuracy of the technique in profiling amino acids from blood spots have previously been demonstrated in diagnosis of such diseases as PKU, maple syrup urine disease, and homocystinuria (29)(30)(31).
The introduction of electrospray tandem mass spectrometry (ESI-MS/MS) to this area of biochemical diagnostics has offered a robust and more sensitive alternative to FAB-MS/MS (32). Automated ESI-MS/MS is specific and accurate, with real potential as a high-throughput method of screening for many IEMs (33)(34). Moreover, the technique has been successfully applied to prenatal diagnosis of several organic acidemias from amniotic fluid and to postmortem diagnosis from bile spots (35)(36). Our objectives in the current study were to develop and evaluate a microplate-based batch method for preparing blood-spot samples, and to develop a computer algorithm for automated flagging of abnormal metabolic profiles by using MS/MS-derived diagnostic parameters for amino acids and acylcarnitines in newborns.
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Materials and Methods |
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chemicals
Acetyl-[d3-methyl]carnitine,
propionyl-[d3-methyl]carnitine,
octanoyl-[d3-methyl]carnitine, and
palmitoyl-[d3-methyl]carnitine were generous gifts of
David Millington (Duke University, Durham, NC).
[d3-Methyl]carnitine,
1-[13C,15N]glycine,
alanine-2,3,3,4-d4, valine-d8,
leucine-5,5,5-d3, methionine-d3,
ornithine-3,3,4,4,5,5-d6,
phenylalanine-ring-d5, and tyrosine-ring-d4
were purchased from Cambridge Isotope Labs. (Woburn, MA). Butanolic HCl
was prepared by saturating dry HPLC-grade 1-butanol with HCl gas for 60
min. HPLC-grade methanol and acetonitrile were purchased from Fisons
Scientific Equipment (Loughborough, UK). "Water for Irrigation" was
purchased from Abbott Labs. (N. Chicago, IL).
blood samples
The blood samples used in this study were collected according to
the guidelines of our Research Council. The samples were taken from
newborn babies in several hospitals throughout Saudi Arabia and from
sick children referred to our metabolic unit. Blood collected by
heelstick was applied to four marked circles on barcoded Guthrie cards
(made of S&S 903 filter paper; Schleicher and Schüll, Dassel,
Germany), allowed to dry, and then returned to our laboratory by mail
or courier service. Upon receipt, the barcode number for each sample
was read by a barcode reader (from PSC, Webster, NY), and the
patient's medical record number and biodata were logged into our
database.
microplate batch preparation
We process samples in batches of 96 (or fewer), in standard
96-well U-bottomed microplates (capacity 300–400 µL per
well). An in-house-developed automated blood-spot puncher punches a
single 3/16-in. (~4.75 mm) blood spot from each Guthrie card directly
into the individual wells of a 96-well microplate. An in-house software
program written in Microsoft Visual Basic, together with a PSC barcode
reader, is used to read the sample ID codes and to map them to the
plate positions as each blood spot is punched. To allow for tracking in
our system, a file name is given to each batch (microplate).
We prepare the samples as previously described (33), but with some modifications. To the blood spot punch in each well we add a methanolic solution (100 µL) containing known concentrations of stable isotope-labeled standards of the following compounds: 1000 pmol each of glycine and alanine; 250 pmol each of valine, methionine, and phenylalanine; 500 pmol each of leucine and tyrosine; 221 pmol of ornithine; 125 pmol of carnitine; 60 pmol of acetylcarnitine; 6 pmol of propionylcarnitine; 6 pmol of octanoylcarnitine; and 12.5 pmol of palmitoylcarnitine. We dispense the methanolic solution with an Eppendorf Repeater 4780 pipette (Eppendorf-Netheler-Hinz, Hamburg, Germany), which uses disposable 1.25-mL "combitips" to deliver several 100-µL aliquots from a single filling. The microplate is covered with aluminum foil, and the samples are extracted for 30 min at 4 °C. The methanolic extracts are then transferred with an 8-tip multipipettor to identical positions in a hexane-resistant vinyl U-bottomed microplate (200-µL capacity per well; Costar, Cambridge, MA). The methanol is removed by heated evaporation at ~50 °C under an electrical hot-air blower. To the residue in each well we add 50 µL of butanolic HCl, again using the Repeater device, and seal the microplate with self-adhesive tape (from Manco, Westlake, OH); derivatization is completed by heating at 65 °C for 20 min. Excess butanolic HCl is removed with the hot-air blower, and the whole plate is washed by immersion in hexane for 1 min; we then shake the hexane out of the wells by tapping the inverted plate on a hard surface and let the plate dry at room temperature. We reconstitute the final residues in 125 µL of mobile phase (see below), again covering the plate with aluminum foil (to prevent evaporation), and place the plate in the autosampler tray for injection into the mass spectrometer.
sample introduction and mass spectrometry
We use a Model PU-980 HPLC pump and a Model AS-950 autosampler
(both from Jasco International Co., Tokyo, Japan) for solvent delivery
and automated sample introduction. The injector outlet port is
connected via a length of fused silica to the inlet of the
electrospray-equipped VG Quattro triple-quadrupole mass spectrometer
(VG Biotech, Fisons Instruments, Altrincham, UK).
Acetonitrile:irrigation water (80:20 by vol) is used as mobile phase
and to flush the autosampler syringe between injections. The
autosampler is fitted with a 20-µL loop and is programed to overfill
the loop (fill volume 26 µL) directly from each well. For all our
routine analyses, the HPLC pump is programed to give a flow rate of 17
µL/min. Injections are made at 2.7-min intervals, a contact closure
signal being generated at injection to start the event cycle of the
HPLC pump. At 0.3 min after injection, the pump gives a contact closure
output, which starts the mass spectrometer's data-acquisition
sequence. This postinjection delay allows time for the sample to reach
the ion source. The pump is programed to momentarily increase the flow
rate to 1.0 mL/min at 2.4 min after injection. This flow surge at the
end of the injection cycle serves to wash the sample loop and clears
any residual material from the electrospray interface of the mass
spectrometer. The remaining 0.3 min of the cycle allows reequilibration
of the liquid flow rate to 17 µL/min before the next injection. The
electrospray source temperature is maintained at 120 °C, and the
pressure of argon gas inside the collision cell is adjusted to 0.4 Pa.
Nitrogen from a cylinder, regulated to 101
kPa, is used as
nebulizing gas, and compressed air from the in-house supply, regulated
to 300 kPa, is the bath gas. We set the resolution of both mass
spectrometers, MS1 and MS2, to give peak widths of 1.2 Da at 15%
height and set the instrument's ion counting threshold to 5.
We use a program developed in-house to retrieve the plate map from the centralized database and to transpose the data into a compatible format for use as a sample list for the mass spectrometer's Masslynx software. The program places the formatted list onto the Windows clipboard of the mass spectrometer's PC for later pasting into the Masslynx sample list editor. A project file created in the Masslynx software is given the same unique name as that of the plate. The plate map list is pasted (from the clipboard) into the sample list editor of the project file and saved.
For all routine work, we programed the mass spectrometer to acquire each patient's sample as a separate data file in the multichannel analysis mode. The following 1.9-min sequence of four MS/MS positive-ion scan functions is initiated by a contact closure from the HPLC pump: (a) free carnitine and acylcarnitines (0.00–1.00 min): precursor ions of mass 85, from 215 to 500 Da, scan time 3.0 s, cone voltage 28 V, collision energy 25 eV; (b) amino acids (1.00–1.45 min): neutral loss of 102 Da from 125 to 300 Da, scan time 0.95 s, cone voltage 20 V, collision energy 10 eV; (c) amino acids (1.45–1.65 min): neutral loss of 119 Da from 150 to 270 Da, scan time 0.75 s, cone voltage 20 V, collision energy 25 eV; and (d) amino acids (1.65–1.90 min): neutral loss of 56 Da from 120 to 155 Da, scan time 0.4 s, cone voltage 20 V, collision energy 8 eV.
At the end of the 1.9-min data-capture cycle, the file name for the next sample is loaded, and the mass spectrometer is reinitialized to receive a new acquisition start signal from the HPLC pump. The time difference between the injection intervals of 2.7 min and the mass spectrometer acquisition time of 1.9 min allows for purging the system, injecting the sample, and letting the sample reach the ion source of the mass spectrometer.
evaluation of microplate batch process
We prepared six microplates (576 samples) from stored blood spots,
including >60 samples from known abnormal cases. Most of the prepared
samples were analyzed twice to generate sufficient data for 1000
consecutive injections. Only for this experiment did we deviate from
our routine conditions (see above) to use a faster flow rate (constant
at 40 µL/min) and a faster injection cycle (one sample every 1.3
min). Also, the electrospray source temperature was increased to
150 °C, to accord with the greater flow rate of the solvent. For
recording multiple sample results in an easily visible format, we used
an 8-s repetitive cycle containing four scan functions to generate a
single continuum data file for acylcarnitines, two groups of amino
acids, and free carnitine. The time allocated to each function within
the cycle was apportioned according to relative ion intensity
considerations within the analyte groups monitored (33).
computer algorithm for data processing
Selecting a set of control samples.
The parameters to be
used by the algorithm were selected by comparing the metabolic profiles
from several known cases of organic acidemias and amino acid disorders
with the profiles from the control samples. A large set of data files
(n = 1100) from our newborn population was chosen for establishing
control cutoff values for the selected parameters. The criteria for
this sample set were as follows: (a) samples from newborn
infants who had a minimum birth weight of 2.01 kg; (b)
samples analyzed in <72 h from time of blood collection; and
(c) data files designated, after manual scrutiny by expert
analysts, as "not remarkable."
Algorithm design and architecture.
We used a specialized
data-processing algorithm, Computer-Assisted Metabolic Profiling
Algorithm (CAMPA), to process the batches of MS/MS data files acquired
in the routine mode of analysis. Written in Microsoft Visual Basic,
CAMPA is based on our previously published automated key strokes
(macro) for data smoothing, measurement, and hardcopy (33)
and is designed to allow automated flagging of abnormal profiles.
Upon execution, table definitions and parameter values needed for the analysis are downloaded from three primary tables, all of which are held in a centralized Access Database. The function of each table is as follows:
1) Acquired functions of diagnostic interest (FDI). This table contains user-defined minimum thresholds for the measured base peak intensity (intensity of the highest peak in the spectrum) of each acquired scan function.
2) Masses of diagnostic interest (MDI). This table, used to define the masses of diagnostic interest for each of the acquired functions, contains the molecular masses corresponding to free carnitine, all acylcarnitines known to be of diagnostic value, all amino acids detectable by this method, all stable isotope-labeled internal standards, and some of the acylcarnitine peaks observed after medication (e.g., phenylacetylcarnitine and benzoylcarnitine, which result from using phenylacetate and benzoate mixture in urea cycle disorders). By using user-defined flags, we can designate some of these masses as "must find" masses. The table also includes (M + 1) Da isotope masses, corresponding to the more abundant peaks observed in the normal profile for acylcarnitines.
3) Peak ratios of diagnostic interest (RDI). All diagnostic peak ratios are defined in this table, along with maximum (and some minimum) cutoff values for each ratio. Blood concentrations are calculated for analytes for which stable isotope internal standards have been included, by using peak-height ratios and a concentration factor defined with each record. Cutoff values for these ratios have been defined in terms of a maximum and minimum molar concentration value. Because isotope-labeled analogs are not included (or available) for several analytes, peak-height ratios are defined relative to internal standard peaks within a similar mass range. Height ratios of certain pairs of analyte peaks are also defined. The cutoff thresholds used are expressed as maximum absolute values for each diagnostic ratio. The denominators of all calculated ratios correspond to masses designated "must find."
Once these three tables have been downloaded, the user is prompted to specify profile printout requirements. Available options allow printing of either all sample data, only sample data designated "abnormal," only sample data designated "low quality," or the combination of the latter two (all data designated "abnormal" or "low quality"). Database storage of all calculated diagnostic information for each sample can also be specified. To permit unattended overnight data acquisition and processing, one can select a timer function that allows the algorithm run to be set up for data not yet acquired. In this case, the user is asked to specify the complete file path specification of the data directory, together with the delay interval that should elapse before processing begins. If the timer function is not utilized, the data files can be selected individually or in highlighted groups by using a custom dialog window.
Automated processing of MS/MS data.
The Masslynx
operating and processing software provided with the instrument is
driven by automated key strokes to access data-processing functions
provided in this software. Data smoothing by successive iterations of a
moving median algorithm (peak-width parameter = 0.4 Da) on
convergence serves to reduce the intensity of narrow data spikes to the
background value. A second smoothing step, based on a Savitzky–Golay
algorithm (peak-width parameter = 1.1 Da), restores a smooth
gaussian shape to the square-edged peaks produced by the moving median
smoothing algorithm (33). After background values are
subtracted from the smoothed spectrum, the absolute intensity (height)
of each peak is measured. Peak masses are assigned to the calculated
centroid (point at which areas are equal on either side of it) of the
upper 50% portion of each peak. The measured masses (to 4 decimals),
and corresponding absolute peak intensities, are presented in a
numerical table within Masslynx. This table is automatically copied to
the Windows clipboard and then retrieved for arithmetic processing
within CAMPA. The program applies a negative mass shift of 0.3 Da to
all the measured mass values in the data array, rounds the shifted
masses to integer values, and sorts and searches the data array for
instances where two or more peaks are listed with the same nominal
mass. These duplicate mass entries do occur on occasion, despite the
smoothing methods described above. In case of duplicates, the entry
with the greater intensity is retained and the remaining entry(ies) is
discarded.
Data analysis.
Subsequent to the capture of the
processed data for the file under inspection, a four-step analysis
phase is initiated:
1) The base peak intensities for each of the four profiles are calculated from the captured data tables and compared with the minimum threshold values, which have been loaded from the FDI table. A "low quality" flag is set for sample data having a base peak intensity below the threshold value for any function.
2) The captured data array is searched for all of the masses defined in the MDI table. When found, these masses are moved into working fields within the MDI table. If none of the masses designated "must find" are found in the data array, the sample data are flagged "low quality." The acylcarnitines section of the data that have not been moved in the array is then searched for remaining peaks (not defined in the MDI) for which the intensity is >5% of the base peak height. Any such peaks, if found, are listed as "exception masses" in the output file corresponding to that sample.
3) Peak-height ratios are calculated from the data in the working fields of the MDI for all peak ratios defined in the RDI. Concentration values can then be calculated and threshold testing performed on the calculated peak ratio or concentration value, as defined in the RDI. "Abnormal" flags are set for sample data giving values outside the defined thresholds.
4) The output log file is checked for any "low quality" or "abnormal" flags. Depending on the initial printout options selected, hard copies of the smoothed analog profiles are automatically generated by the CAMPA keystroke-driven Masslynx software. Finally, a formatted report is printed, listing the samples flagged as "abnormal" or "low quality" and detailing the reasons for each flag. Also listed are those samples flagged to indicate detection of "exception masses."
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Results |
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microplate batch process and sample throughput
In the new microplate batch process we developed, 96 samples per
batch could be prepared in ~2.5 h, a major improvement over
previously published methods (31)(32)(33), which use three
vials (or tubes) per sample and require labor-intensive labeling,
capping, uncapping, and individual transfer of extracts between vials.
In a qualitative experiment we estimated the biological load that the
electrospray interface can accommodate without loss of data quality.
Fig. 1
shows the first (A) and last (B) 200 min of the acylcarnitines
profile from a sample run comprising data from 1000 blood-spot
extracts, prepared by the microplate batch process for one
4.75-mm-diameter punch from each blood spot. Because this experiment
was not practical at our routine injection interval time of 2.7 min
(i.e., total run time would have been 45 h), we used a faster
injection cycle and flow rate. Visual examination of the profiles
indicated very little loss in sensitivity between the beginning and end
of this 1300 min (~22 h) run. Profiles for the other functions
monitored simultaneously were of equal quality (data not shown). Fig. 2
shows the abnormal acylcarnitines profiles obtained for a
patient with glutaric acidemia type-II (GA-II), from whom a processed
blood spot was injected at 494 min and repeated at 1258 min into the
run. Comparison of the two profiles illustrates the high consistency
(reproducibility) in spectral quality achieved with our method
throughout this protracted multiple-sample run.
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determination of newborn population reference values
The algorithm developed (CAMPA) was initially used to process and
quantify selected parameters from a group of >1000 profiles, which had
been acquired individually as separate data files under our routine
acquisition conditions (multichannel analysis mode and 2.7-min cycles)
and classified as "normal" by manual scrutiny. We made no
assumptions as to the nature of the distribution of the analyte
concentrations or peak ratios, although for most the mean and median
were very close. To determine cutoff values for these analytes, we used
the percentile method (38). Accordingly, the values
obtained for each analyte were ranked, and the 0.5th and
99.5th percentiles were calculated. The 99.5th
percentile was set as the high limit for each of the parameters. For
some key metabolites a lower concentration limit, corresponding to the
0.5th percentile, was also used. The cutoff values obtained
for amino acids and acylcarnitines were then plugged into CAMPA.
Table 1
shows the blood concentration and cutoff values for key amino
acids in our newborn population. The mean blood concentration for
phenylalanine in our study was 72 ± 21 µmol/L, in agreement
with literature values for newborns both by FAB-MS/MS and by HPLC
(29)(39)(40). A phenylalanine
cutoff value of >129 µmol/L (99.5th percentile) is very close to the
internationally recognized value of 120 µmol/L (2 mg/dL) used in many
PKU (or hyperphenylalaninemia) screening programs
(10)(12).
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The mean concentration for tyrosine was 100 ± 46 µmol/L, also similar to the 82 ± 20 µmol/L reported in newborns (39). The mean concentration for methionine was 28 ± 10 µmol/L, quite close to the 31 ± 8 µmol/L determined by HPLC for 11-day-old infants reported by Scott et al. (39), and an upper cutoff value of 59 µmol/L for blood methionine obtained in this study agreed favorably with the 67 µmol/L (1 mg/dL) used in screening for isolated hypermethioninemia and homocystinuria in some programs (13)(31).
Valine concentration in our newborn population was 116 ± 36 µmol/L, compared with 111 ± 24 µmol/L determined by HPLC (39) and with 131 ± 58 µmol/L by FAB-MS/MS (30). Our value of 103 ± 33 µmol/L for leucine (+ isoleucine) was less than the literature values reported for the other methods (30)(39). The mean blood concentration of glycine, however, 402 ± 111 µmol/L, was higher than the 191 ± 96 µmol/L reported by Scott et al. (39).
Table 1
also presents average abnormal concentrations and ranges for
diagnostic metabolites in known cases as they pertain to certain amino
acid disorders. The data for some disorders are combined when the
biochemical marker is the same, e.g., for
homocystinuria/hypermethioninemia and for tyrosinemia type I/type II.
In tyrosinemia type I, methionine may also be substantially increased;
however, both biochemical markers are not sufficiently diagnostic for
the disease (33).
Some other diagnostic parameters based on peak-height ratios of pairs
of analytes are also included in Table 1
. The peak ratio of citrulline
to tyrosine acquired in the scan function for neutral loss of 119 Da
was extremely useful for detecting several cases of argininosuccinic
synthetase deficiency and argininosuccinase deficiency
(33). A peak ratio of the less abundant deammoniated butyl
citrulline (m/z 215) to phenylalanine (in the neutral loss
of 102 Da scan function) was also useful for argininosuccinic
synthetase deficiency. Furthermore, we have diagnosed two cases of
hyperprolinemia by measuring the peak-height ratio of proline
(m/z 172) to phenylalanine. However, proline was also
somewhat increased in some cases of liver dysfunction. Other ratios of
less diagnostic power were those for pyroglutamic acid (pipecolic;
m/z 186) to phenylalanine, used in the diagnosis of
pyroglutamic acidemia or pipecolic acidemia (33).
Most of the diagnostic parameters obtained from acylcarnitines were
peak-height ratios relative to a known amount of an internal standard
(usually d3-octanoylcarnitine,
d3-C8), or peak-height ratios of some analyte
pairs. Table 2
shows a description of each of these diagnostic ratios and
provides a comparison between the upper cutoff value and the average
abnormal value for each marker as they pertain to different disorders.
The mean blood concentration of free carnitine in our unaffected
newborn population was 22 ± 8 µmol/L, which is intermediate
between concentrations of 15 ± 4 µmol/L in plasma isolated from
cord blood and 34 ± 5 µmol/L for plasma from children (ages 2
months to 5 years) as reported by Bhuiyan et al. (19). The
lower and upper cutoff values for free carnitine determined by our
method were 9 and 59 µmol/L, respectively. This parameter is not
included in Table 2
because it varies significantly with disease,
metabolic status of the patient, and treatment. Most of the diagnostic
parameters, however, showed a clear distinction between the abnormal
value obtained from patients with the metabolic disorder and the upper
cutoff value from the population of unaffected newborns.
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testing the discriminative power of campa
Two retrospective experiments were carried out to verify the
performance of CAMPA and to quantify the specificity and sensitivity of
the algorithm before introducing it into unchecked daily service in our
laboratory. The first test consisted of 559 MS/MS data files that had
been compiled in one directory on the processing PC. Among these were
119 "abnormal" profiles obtained from patients with known metabolic
diseases who had been diagnosed from MS/MS results and who exhibited
clinical symptoms; many of these samples were from patients undergoing
treatment at the time of sample collection. The remainder (440)
were "normal" profiles from the neonatal screening samples. The
second test involved a larger data set of 1151 files: 147
"abnormal" files (including the previous 119 files) and a new batch
of 1004 "normal" data files from neonatal screening samples. CAMPA
was applied to both data sets, and summary reports were generated and
compared with previously obtained manual findings. Collectively, the
results indicated that the sensitivity of the algorithm was 100% and
the weighted average cumulative specificity for the two tests was
83.1% (Table 3
).
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A flow chart (Fig. 3
) summarizes the processes that occurred when CAMPA was launched
for a selected data set. Fig. 4
shows an example of the output of the computer algorithm, i.e.,
an abnormal acylcarnitine spectrum for a case of medium-chain acyl-CoA
dehydrogenase deficiency diagnosed by this technique in neonatal
screening samples. The output report for this sample, as determined by
CAMPA, is pasted to the spectrum. Basically, four abnormally increased
species are evident in the spectrum: octanoylcarnitine (C8;
m/z 344), hexanoylcarnitine (C6; m/z
316), decanoylcarnitine (C10; m/z 372), and
decenoylcarnitine (C10:1; m/z 370)
(33)(41)(42). As presented in the
spectral data and the abnormality details, the most outstanding
increase was in the concentration of C8, which was 22-fold
above the upper normal value in our newborn population.
C6, C10, and C10:1
were also above the respective threshold values.
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Discussion |
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Our main objective in the current study was to take further steps toward establishing ESI-MS/MS as a robust, high-sample-throughput, and efficient neonatal screening method. Our first goal was to increase sample throughput by simplifying sample preparation. We converted the sample preparation to a more efficient and cost-effective batch-type process by using the convenience of 96-well microplates. Automated punching and microplate mapping eliminated the tiresome process of labeling individual sample vials and writing lists of sample names. The inherent possibilities for sample confusion within these processes have accordingly been substantially reduced. Generally, two technicians can now prepare six microplates (576 samples) in a regular working day. In addition, the use of one 4.75-mm (7 µL) blood-spot punch reduced by half the biological load of sample injected into the ion source of the mass spectrometer. This made it practical to inject 500 samples per day into an instrument for our routine cycle of 2.7-min intervals between injections (total: 1350 min or 22.5 h) and to acquire each sample as a separate data file. We foresee that the whole process could be fully automated by using modified microplate robotics, which might further reduce the cost of labor and improve precision.
Data interpretation by manual scrutiny is a labor-intensive and subjective process requiring considerable experience and expertise. The algorithm, CAMPA, was developed to transform the interpretation process into an objective, efficient, quantitative, and automated process. Designed as a flexible program, CAMPA is totally configured via the parameter setup in the database tables. This allows an expert user to change the concentrations of the internal standards added during sample preparation, whereby all formulas for metabolite concentrations are then automatically adjusted. In addition, CAMPA can easily allow addition, or elimination (or inactivation), of various markers as more information is acquired from new metabolic cases and from larger data sets for our newborn population.
Several criteria were extremely important for achieving the goals for which the algorithm was developed. The first criterion was to provide the algorithm with high-quality MS/MS data, free from detector artifacts that might otherwise be designated as metabolite peaks, and with no errors in mass assignments. In this regard, introduction of the combined median and Savitsky–Golay smoothing techniques proved useful for eliminating detector spikes from the raw spectra, nullifying the potential effects of these spikes on the relative heights of the diagnostic peaks, without substantially affecting peak width. In our previous work with acylcarnitines, we noted that integer mass assignments were sometimes in error by a 1-Da excess; the Masslynx software had generated these integers by rounding off measured values (i.e., 347.50 would become 347 and 347.51 would become 348). Instrument considerations, peak asymmetry, and low ion counts can produce a measured mass accuracy error of ~± 0.2 Da in the longer-chain acylcarnitines. Considering that the calculated monoisotopic mass of the butyl ester of palmitoylcarnitine is 456.4 Da, rounding measured values might occasionally assign the ester an integer mass of 457. To overcome these inconsistencies, we used a "Delta" mass shift facility (provided in the Masslynx peak annotation software) to apply a 0.3-Da negative mass shift to all measured masses before rounding them off. In designing the analytical mechanism of CAMPA, we decided to incorporate a delta mass shift, similar to that used in the printout annotation, within the data-processing structure of CAMPA. This process has also been shown to give correct integer values for masses in the amino acids profiles. Mass entries in the MDI and RDI tables were also made as integer values. The unique numerical values for diagnostic masses and concurrence with printout annotation provided by this system have simplified interpretation of CAMPA output and discussion of profile abnormalities.
Another important criterion for CAMPA was to provide it with adequate
parameters to be tested. Successful selection of these parameters was
possible because of the large number of metabolic cases in our files
and the wealth of data available in the literature. Selection of
flagging markers for amino acid disorders was straightforward, usually
depending on the increase of one or more amino acids, e.g., methionine
in hypermethioninemia/homocystinuria, and leucine (+ isoleucine) and
valine for maple syrup urine disease. In some diseases, the variability
of the data could be reduced by establishing markers as a ratio of one
amino acid to another, e.g., the phenylalanine/tyrosine ratio for
hyperphenylalaninemia, and methionine/leucine for hypermethioninemia
(13)(15). The situation is not as simple for
organic acidemias and fatty acids oxidation defects. Although for some
diseases a certain metabolite is the key in making a preliminary
diagnosis, e.g., increased glutarylcarnitine in glutaric acidemia type
I (GA-I), for most of these disorders several metabolites were found to
be increased. For example, in GA-II (Fig. 2
), in medium-chain acyl-CoA
dehydrogenase deficiency (Fig. 4
), and in long-chain (or
very-long-chain) acyl-CoA dehydrogenase deficiency, several
acylcarnitines were increased above normal values
(33)(41)(42). In addition, for
most of these disorders, measurement of free carnitine is important in
the initial blood sample and in posttreatment follow-up samples.
Accordingly, many parameters had to be selected and cutoffs established
for these disorders (see Table 2
). Unlike the amino acids, the
concentration of some of the diagnostic acylcarnitines in normal blood
samples is often below the detection limit of the method, e.g.,
glutarylcarnitine (C5-dicarboxylic), diagnostic for GA-I;
3-methylglutarylcarnitine (C6-dicarboxylic), diagnostic for
3-hydroxy-3-methylglutaryl-CoA-lyase deficiency; and tiglylcarnitine
(C5:1), which is of diagnostic value in ß-ketothiolase
deficiency (26)(33). For this reason, we
applied lower cutoff limits only to those metabolites that were
consistently present in spectra of normal samples, such as free
carnitine, acetylcarnitine, and palmitoylcarnitine.
Determination of population-based cutoff values from a large number of
normal samples was another important criterion toward achieving high
reliability in discernment of abnormal profiles. Although CAMPA is
being used for both selective and neonatal screening for metabolic
diseases, the population selected for analysis to determine cutoff
values was normal (unaffected) neonates. The decision to use values
from newborn blood samples was made because of the availability of a
large number of samples from this population, and because this is our
target group for a neonatal screening program. The ability to use CAMPA
to calculate and store all values obtained from a large data set
(n = 1100) allowed us to establish reliable cutoff values for the
selected markers. For the most part, values obtained for key blood
metabolites by ESI-MS/MS, particularly for the amino acids, are very
similar to those obtained by conventional techniques. The success of
the algorithm has been evident in the absolute sensitivity of CAMPA for
flagging cases with known metabolic disorders. As for the difference
between the sensitivity and the specificity of the test (<17%), we
found that variability in the acylcarnitine values was responsible for
>85% of "false flagging." In particular, propionylcarnitine and
butyryl (or isobutyryl)carnitine ratios just above the respective
cutoff values were responsible for ~25% of the falsely flagged
samples, whereas in reality these minor increases are of no
pathological significance. Of the falsely flagged data, <5% was due
to borderline increase of one or more of the key amino acids such as
methionine, leucine (+ isoleucine), alanine, glycine, and
phenylalanine. The remaining 10% were the result of borderline low
values for one or more of these amino acids. In most falsely flagged
data files, a single parameter was usually the reason the flag was set.
In most "truly abnormal" data files, however, multiple flags were
set, and the values obtained for concentrations or ratios were
noticeably higher than the cutoff values (see Tables 1
and 2
).
Routinely, the analyst examines the report detailing the reasons for
flagging each data file and visually inspects the spectra for these
files, thereby permitting an accurate diagnosis. He or she then decides
whether to repeat analysis of the sample or to annotate it as "not
remarkable." Because of the severity of many of these diseases, a
report would immediately be sent out to the clinician, for any patient
whose profiles were clearly abnormal, as determined by CAMPA, followed
by expert concurrence. Also, to assure reproducibility of the initial
results, the sample would be reanalyzed in the next batch of samples.
In developing a new screening method or program, the probability of both false-positive and false-negative results should be rigorously tested. A high rate of false-positives will both increase the cost of the screening program and put an emotional burden on the parents, when they are requested to submit repeat samples. As pointed out by Chace et al. (31), and by the work presented here, the specificity and accuracy provided by MS/MS over conventional screening methods should reduce the rate of false-positives and therefore reduce the number of repeat samples. False-negatives are even more costly: They involve missing cases of possibly treatable disorders and could seriously undermine the screening program. In this study, the calculated sensitivity for the method was based on the results for known metabolic cases and, to the best of our knowledge, we have not encountered a false-negative case. However, additional time and more testing are required to further ensure the reduction or elimination of false-negatives. In this regard, development of collaborative interlaboratory quality-assurance programs appears necessary to further validate the method.
In conclusion, the development and use of CAMPA and the microplate batch process has allowed all laboratory personnel in our metabolic unit to proceed with a batch of samples from initial sample registration through production of a final printed output report. The introduction of CAMPA has provided our expert analysts with a powerful quantitative tool for detecting abnormal profiles and has substantially decreased the time spent in manual scrutiny of multiple spectra for each patient's sample. These new facilities have given the unit a high-throughput capacity, allowing analysis of hundreds of samples on a daily basis. We believe that these advances in metabolic profiling methodology, and the resulting cost-effectiveness per sample, make ESI-MS/MS the method of choice for establishing neonatal screening programs for an important group of metabolic diseases. In this regard, our laboratory has recently started a pilot neonatal screening program for this category of diseases in Saudi Arabia.
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