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1
Neo Gen Screening, 110 Roessler Rd., Pittsburgh, PA 15220.
2
Centers for Disease Control and Prevention (CDC),
Newborn Screening Quality Assurance Program, Atlanta, GA 30341-3724.
3
Duke University Medical Center, Mass Spectrometry
Facility, Research Triangle Park, NC 27709.
a Author for correspondence. Fax 770-488-4831; e-mail whh1{at}cdc.gov
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: To each of six portions of a pool of washed erythrocytes suspended in serum we added Phe (0–200 mg/L), Leu (0–200 mg/L), Met (0–125 mg/L), Tyr (0–125 mg/L), and Val (0–125 mg/L). Six-pool sets (1300) were prepared, dried, and packaged. We used isotope-dilution mass spectrometry to estimate the endogenous amino acid concentrations of the AARMs and validate their final amino acid concentrations. We used additional tandem mass spectrometry analyses to examine the homogeneity of amino acid distribution in each AARM, and HPLC analyses to evaluate the stability of the amino acid contents of the AARMs.
Results: The absolute mean biases across the analytic range for five amino acids were 2.8–9.4%. One-way ANOVAs of the homogeneity results predicted no statistically significant differences in amino acid concentrations within the blood spots or within the pools (P >0.05). Regression slopes (0 ± 0.01) for amino acid concentrations vs storage times and their P values (>0.05) showed no evidence of amino acid degradation at ambient temperatures, 4 °C, or -20 °C during the intervals tested.
Conclusion: The validation, homogeneity, and stability of these blood spots support their use as a candidate national reference material for calibration of assays that measure amino acids in dried-blood spots.© 1999 American Association for Clinical Chemistry
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effective screening of newborns with the use of blood-spot samples collected from newborns soon after birth, combined with diagnostic studies and treatment, helps prevent mental retardation and premature death. Most newborn screening laboratories use DBS calibrators for their newborn screening tests. These whole-blood calibrators for DBS methods control for sample-elution effects and other effects contributed by the dried-blood-paper matrix. In most methods, liquid calibrators do not appear to adjust for the matrix contributions and sample elution of analytes measured in DBSs. In a pilot study among four laboratories that used identical aqueous-based calibrators and identical assay methods for measurement of amino acid content in sets of DBS samples and aqueous-based samples, we found that (a) the analyte recoveries in the assays with aqueous-based calibrators showed an interlaboratory variability with DBS samples that is not seen with the corresponding aqueous-based samples, thus confirming the need for DBS calibrators that go through the same blood-spot extraction process as the unknown DBS samples; and (b) the use of common accuracy-based calibrators and identical methods substantially reduces interlaboratory variability (10). These factors support the position that accuracy-based DBS calibrators for amino acid DBS tests are needed. To meet this identified need, the Newborn Screening Quality Assurance Program operated at the CDC in Atlanta, GA produced 1300 six-pool sets of multianalyte DBS candidate amino acid reference materials (AARMs) (11) by dividing a single batch of mixed-donor blood of measured hematocrit into six pools; enriching the pools with pure amino acids to cover the usual analytic ranges of Phe, Leu, Met, Tyr, and Val; and dispensing 100-µL spots of the blood pools onto Schleicher & Schuell (S&S) Grade 903 blood-collection paper.
The development of certified reference materials is the first element in any comprehensive effort to standardize an analytic assay system. The variety of assays for amino acids must be standardized to ensure the comparability of data and the accurate interpretation of presumptive clinical assessments from laboratory measurements, especially when small changes from physiologic cutpoints may be important predictors of disease risk. When the concentrations of the assay calibrators are certified by use of an IDMS method, the accuracy of measured values is attainable among laboratories through the standardization process. Primary standards, purified amino acids, are not suitable in most newborn screening DBS methods because matrix characteristics are not adequately managed (10); therefore, a certified blood-spot product is essential for assay calibration of newborn screening methods. Here we provide the data for such a candidate DBS reference material.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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stock calibrator solutions for aarm whole-blood pools
The candidate reference materials were enriched with two stock
calibrator solutions: solution A, containing Met, Tyr, and Val, each at
a concentration of 5 g/L in dilute HCl; and solution B, containing Phe
and Leu, each at 5 g/L in deionized water.
materials for the tandem ms analyses
Stable isotopes of
[2H5]Phe,
[2H3]Leu,
[2H3]Met,
[2H4]Tyr, and
[2H8]Val were purchased
from Cambridge Isotopes as mass spectrometry (MS) internal standards.
These internal standards were of both high chemical and isotopic
purity, >99% as certified by the isotope manufacturers. Stock
solutions of the internal standards were prepared in deionized water
and HCl from J.T. Baker, and aliquots were added to whole-blood samples
for IDMS analyses or to a methanol-deionized water (1:1, by
volume) DBS sample-extraction solution for other tandem MS
(MS/MS) analyses. High-purity methanol from Burdick and Jackson was
used for sample extraction, butanolic-HCl (3 mol/L) from Regis was used
for derivatizing the amino acids, and glycerol and sodium octyl sulfate
from Sigma were used to reconstitute the samples for MS analyses
according to methods published previously (3)(4)(5).
materials for hplc analyses
3,5-Dibromo-L-tyrosine
[(Br)2Tyr], ornithine (both from Sigma), and
200 proof ethanol (Pharmco) were used to prepare the blood-spot
extraction solution for HPLC analyses; Amino Acid Standard H (lot no.
891207087) from Pierce was used to calibrate the HPLC analyses. To
perform the HPLC analyses, we used a WatersTM
AccQ.FluorTM Reagent kit (cat. no. 52880) and
Eluant Concentrate (cat no. 52890), reagent-grade water and
acetonitrile (Sigma), and Nova Pak column guards and an AccQ.
TagTM analytical column (cat. no. 52885; Waters).
preparation of aarm dried-blood spots
Recently collected blood units of insufficient quantity for
transfusion were washed with three portions of saline to remove
anticoagulants and the buffy coat. After centrifugation and removal of
the last saline wash, the combined red cells were reconstituted to a
hematocrit of 57% ± 0.5% with pooled human serum that had been
clarified by serial filtration through filters with pore diameters of
1.2, 0.8, 0.65, 0.45, and 0.22 µm. This blood batch was divided into
portions for enrichment with Phe and Leu at 0, 40, 80, 120, 160, and
200 mg/L blood and Met, Tyr, and Val at 0, 25, 50, 75, 100, and 125
mg/L blood. The liquid added during enrichment was sufficient to reduce
the hematocrit to 53% ± 0.5%.
After enriching the whole-blood pools with the amino acids, adding sufficient blood to bring them to volume, and mixing them thoroughly, we refrigerated them overnight. To minimize the effects of fluctuations in room temperature and heat transfer from the magnetic stirrer on which the blood pools were placed, we brought the pools to the temperature of a running water bath and left them in the bath during the dispensing process. Micromedic (ICN Biomedicals) Model 25,000 piston-driven automatic pipettes were used to dispense the blood. These pipettes were gravimetrically calibrated to dispense the six pools of whole blood with intact red cells in 100-µL portions onto S&S Grade 903 (lot no. W941) filter paper placed horizontally on special racks. We dried all blood spots overnight under ambient conditions before transferring them to zip-close metalized plastic bags containing desiccant packets and storing them at -20 °C. Humidity within the containers was maintained at <30% during storage.
preparation of isotope-enriched AARMs FOR MEASURING
ENDOGENOUS AMINO ACID CONCENTRATIONS
A portion of each amino acid-enriched whole-blood pool used to
prepare the AARM blood spots was combined with 40 µL of a solution
containing deuterium-labeled amino acid calibrators to make a final
volume of 5 mL per pool. These whole-blood materials for IDMS analysis
were dispensed onto S&S Grade 903 (lot no. W941) paper, dried overnight
under ambient conditions, and stored at -20 °C in zip-closure
metalized plastic bags containing desiccant packets.
ms/ms analyses
We extracted amino acids from the DBS disks, using a published
procedure (3) modified for use with one 3/16-inch
(~4.8-mm) DBS disk per analysis.
Isotopically labeled internal standards for the IDMS samples were incorporated into the dried-blood samples during preparation; thus, no additional internal standard was added during IDMS sample preparation for MS/MS analysis. However, the AARM blood spots used to assess the homogeneity of amino acid distributions within the DBSs and within the DBS pools did not contain isotopically labeled amino acid internal standards. Internal standards for these MS/MS analyses were added to the methanol extraction solution as described previously (3)(4)(5) and thus did not go through the same extraction process as the amino acids contained in the blood spots.
A Quattro I triple-quadrupole tandem mass spectrometer from Micromass Instruments (formerly Fisons, VG) with a Lab-base data system (Micromass) was operated in the liquid secondary ionization mode [fast ion bombardment, as described previously (4)(5)]. We introduced samples using a static probe at a frequency of ~1 sample/3 min. Neutral loss of 102-Da scan functions were used with a mass range of 125–300 to accumulate data for 1 min as described previously (3)(4)(5). We postprocessed data using a program that assigned quantitative values for each mass of interest and then calculated ion abundance ratios of the masses for metabolites of interest. In this paper, we define ion abundance ratios as the ratio of the ion abundance for the native amino acid divided by the ion abundance for the deuterated amino acid. For example, the ratio of ion intensity of Phe (m/z 222) to the respective internal standard ion intensity of d5-Phe (m/z 227) was determined for each analysis. These ratios can be converted to concentrations by applying correction factors and constants derived from known calibrators and/or calibration curves.
hplc analyses
We used previously described sample extraction (12) and
derivatization (13) procedures, with modified DBS disk size
and sample elution time, to prepare samples for HPLC analysis. Elution
of amino acids from 1/4-inch (6.4 mm) DBS samples into a 700
mL/L ethanol solution containing (Br)2Tyr
internal standard was facilitated by a 10-min sonication. A 20-µL
aliquot of each eluate was combined with 60 µL of AccQ.Fluor borate
buffer and 20 µL of AccQ.Fluor derivatizing reagent, and the
mixture was heated for 10 min at 55 °C.
We used protocols provided by the manufacturer to perform amino acid analyses with a Waters AccQ.Tag HPLC system. The HPLC system consisted of an autosampler and LC Module I pump, a control system with a column heater maintained at 37 °C, an inline degasser, and a 474 scanning fluorescence detector (Waters) set for an excitation wavelength of 250 nm and an emission wavelength of 395 nm. A reversed-phase AccQ.Tag 150 x 3.9 mm C18 column with a solvent system consisting of a three-eluent gradient (AccQ.Tag eluent, acetonitrile, and water) was used at a flow rate of 1.5 mL/min for a sample run time of 19 min. Separation and data reduction were controlled by Millinium® 2010 Chromatography Manager (Waters) software.
determining the endogenous amino acid concentrations of the blood
used for the AARMs
From duplicate IDMS analyses in each of 16 analytical runs, we
determined the mean ion abundance ratio of each of the five amino acids
for every pool in the AARM series. The means of the ion abundance
ratios for each amino acid were plotted as ordinate or
dependent-variable y values against the amino acid
enrichments of the pools (abscissa or independent-variable x
values), and a weighted linear regression analysis of the resulting
plot was used to determine the y-intercept and slope of the
regression line. We calculated the regression analysis weights as the
inverse variances of the MS response measurements at each
concentration. The y-intercepts determined from the Phe,
Met, Tyr, and Val regression analyses were equivalent to the endogenous
concentrations of the blood batch from which the DBS reference
materials were made. The y-intercept determined from the Leu
regression analysis represented the sum of (Leu + Ile) because, as
described previously (5), Leu and Ile have identical masses
and, therefore, are not separated by this MS/MS method. However,
certain assumptions let us closely approximate the sum of the
concentrations of Leu and Ile, and using HPLC we were able to quantify
each of these amino acids (5). We used HPLC analyses to
measure the Leu and Ile fractions in the nonenriched AARM pool; we then
derived the endogenous Leu concentration by multiplying the
y-intercept, determined from IDMS analyses, by the Leu
fraction (0.66), determined from HPLC analyses. We recognize that Leu
values are derived and that this derivation may not necessarily be
applied to every newborn sample. It was important to include these data
for the following reasons: (a) These calculations can be
replicated by other users of MS/MS as described in the validation
article on Leu, Ile, and Val by MS/MS (5), and
(b) it is a reference point for other technologies that
allows comparison between other methods that measure either Leu and Ile
individually or as a total (Leu + Ile) in control or nondiseased
blood spots. We determined the square of the coefficient of correlation
(r2) from each linear regression to
indicate the strength of the among-pool linear relationships.
assigning the target concentrations of the aarm
materials
We defined the target concentration of each amino acid in an AARM
pool as its endogenous concentration plus its enrichment (its added
concentration). We chose this method of setting the target
concentrations on the assumption that amino acid calibrator solutions
could be prepared and added to the blood pools more accurately than the
amino acid content of the DBSs could be extracted and measured.
validating the target concentrations of the
AARMs
The weighted linear regression plots used to establish endogenous
concentrations of the amino acids in the AARMs were also used to derive
their measured concentrations. To correct for the concentration bias
that would be introduced by leaving the endogenous concentration
(y-intercept or b value) on the plot, we
subtracted it from every point along the regression line so that the
linear equation (y = mx + b)
became y = mx. We then divided the measured
ion abundance ratios (y-axis values) by the regression line
slope (m) to derive the measured concentrations (x-axis
values).
To derive the endogenous Ile concentration, we multiplied the IDMS-measured (Leu + Ile) concentration in the nonenriched AARM pool (pool A) by the Ile fraction (0.34), determined from HPLC analyses. We then subtracted the endogenous Ile from the (Leu + Ile) concentrations of all six AARM pools to yield their measured Leu concentrations. Finally, we validated the target concentrations by comparing the assigned target concentrations of the five amino acids with their measured concentrations.
evaluating the homogeneity of analyte distribution within
the aarm blood spots
In a series of 16 MS/MS analytical runs, we measured the ion
abundance ratios of Phe, Leu, Met, Tyr, and Val in north, east, south,
and west disks punched from DBSs on the first and last cards of each
AARM pool and used these ratios in a one-way ANOVA to determine the
probability that disks taken from different locations within the DBSs
contain the same mean amino acid concentrations.
evaluating the homogeneity of analyte distribution within
the aarm production batch
From the 16-run series of MS/MS analyses, we used the amino acid
ion abundance ratios of DBS disks taken from the north position of DBSs
on the first, last, and four intermediate cards of pools C and F in a
one-way ANOVA to determine the probability that disks punched from
different cards within a pool contain the same amino acid
concentrations.
estimating amino acid stabilities in AARMs stored at various temperatures
Strips of blood spots from AARM pool D were stored in zip-closure
plastic bags with desiccant packets and humidity indicator cards in the
dark at 37 °C, ambient temperature, 4 °C, and -20 °C. At
predetermined intervals, strips of DBSs were transferred from
increased, ambient, and refrigerated temperatures to storage at
-20 °C, and strips of DBSs initially stored at -20 °C were
transferred to storage at -70 °C. The humidity of all samples was
controlled to <30% throughout the storage studies. All samples in
each complete time and temperature set were analyzed in duplicate in
each of two HPLC analyses. We used simple linear regression analysis of
measured amino acid concentrations vs storage time to assess the
stability of the amino acids at each storage temperature, and we tested
each regression slope with a statistical t-test to evaluate
whether it was significantly different from zero.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. By-analyte linear regression analyses of ion abundance ratios
determined by IDMS vs enriched amino acid concentration yielded
r2 values >0.98 for all analytes
except Tyr. Visual inspection of ion abundance ratios plotted vs
enriched amino acid concentrations showed an ion abundance ratio for
Tyr in pool E that was much lower than expected. For this reason, we
deleted the analytic results from Tyr pool E and recalculated the Tyr
linear regression to derive the Tyr endogenous concentration and
r2 value that are shown in Table 1
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validation of the target concentrations of the aarm
materials
We compared the target amino acid concentrations with
concentrations measured by IDMS to assess the degree of concordance
between the two sets of concentration values. The absolute mean biases
across the analytic ranges of the amino acids were 2.8% for Phe, 6.4%
for Leu, 4.8% for Met, 3.1% for Tyr, and 9.4% for Val. The Tyr ion
abundance ratio in pool E was much lower than expected. The results of
MS/MS analyses used to assess homogeneity of amino acid distribution
within the DBSs and within the production lots and HPLC analyses (data
not shown) indicated that the spurious Tyr result from IDMS was related
to an error in internal standard addition. The results from the
secondary MS/MS and HPLC analyses indicated a linear relationship among
all Tyr pools and supported the validity of the Tyr target
concentration of pool E.
within-spot homogeneity of analyte distribution in the aarm
blood-spot pools
The P values determined from one-way ANOVAs express the
probability that amino acid concentrations are the same throughout the
blood spot. P = 0.95 indicates a 95% probability that
amino acid concentrations are identical throughout the blood spot;
P = 0.05 indicates a probability of only 5% that amino
acid concentrations are identical throughout the blood spot. Table 2
shows the P values of within-spot distributions for
the five amino acids with which the AARM pools were enriched.
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homogeneity of analyte distribution within the aarm
pools
The P values from the one-way ANOVAs, shown in Table 3
A, are derived from one measurement per card in each of 16
analytical runs. Sixteen measurements were available from each card
included in these MS/MS analyses. The mean ion abundance ratios for Phe
are typical of the card-to-card variability and are shown in Table 3B.
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estimating the amino acid stabilities at
37 oC, AMBIENT TEMPERATURE,4 oC, AND -20 oC
The P values derived from the linear regression
analysis of amino acid concentration vs storage time predict no
statistically significant amino acid degradation during storage at
-20 °C for 1 year and no statistically significant degradation
during the intervals at which the samples were stored at ambient or
refrigerated temperatures with humidity controlled at <30%. (If
P values were >0.05, we concluded that the regression
slopes were not significantly different from zero and that we therefore
had no evidence of amino acid degradation.) All five amino acids showed
significant degradation after storage for 28 days at 37 °C. The
analytic results for Phe shown in Fig. 1
are representative of the thermal stability data observed for
the other amino acids.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because all pools in the AARM series were made from a single batch of whole blood, all contained identical endogenous amino acid concentrations. At low concentrations, such as the endogenous amino acid concentrations in the AARMs, baseline noise can contribute substantially to assay imprecision. To derive more precise estimates of the endogenous concentrations, we used ion abundance ratios of the analytes, determined at multiple concentrations, in linear regression analyses.
Because the molecular ion and its fragment ions for Leu and Ile generated by MS/MS methods are identical in mass, we used HPLC analyses to determine the relative endogenous concentrations of these two isomers and multiplied the MS/MS-generated combined total of (Leu + Ile) by the Leu fraction to obtain the endogenous Leu concentration in the AARM pools. The derived concentrations of Leu, from the total of Leu and Ile concentration, is essential for newborn screening purposes, particularly for MS/MS, which uses a derivation (5) to estimate the total concentration of Leu and Ile. These derived concentrations may be important for other methods as well; therefore, the derived values were computed for this analyte in the DBS reference materials to benefit and inform all users. The endogenous concentrations of the other four amino acids were directly equivalent to the y-intercepts determined from the regression analyses of mean ion abundance ratios vs enriched concentrations. In contrast, the endogenous Phe value for the base-blood pool for production of the European Phe DBS reference material was based on HPLC measurements obtained from multiple laboratories (15).
Because the ion abundance ratio for Tyr in pool E was much lower than expected, and subsequent analyses by other methods indicated an accurate Tyr enrichment of pool E, we omitted the pool E Tyr ion abundance ratio from the linear regression analysis used to determine the endogenous Tyr concentration of the AARM pools and the calculation of the r2 value for that regression line. After the Tyr r2 value was corrected, all r2 values were >0.98, indicating strong among-pool linear relationships for all amino acids.
The endogenous concentrations of amino acids are reflected in the
differences between the enriched and target concentrations of the AARM
pools. Comparison of MS/MS-measured amino acid concentrations with
target concentrations showed concordance between the two sets of
values, thus validating the target concentrations. P values
0.05 would indicate a statistically significant probability, at the
95% confidence level, of nonhomogeneous analyte distribution within
the blood spots. The ANOVAs of ratios of signals from amino acids in
the DBSs to signals from the deuterated amino acid internal standards
added to the extraction solution predicted no statistically significant
differences in amino acid distributions within the blood spots. In
examining the homogeneity of amino acid distributions throughout the
production lots, we found that the P values showed no
statistically significant differences in amino acid concentrations
among cards within a pool. Variability of amino acid recovery from the
AARM pools was not related to card number.
In a recent review of DBS storage stability studies, Therrell et al.
(16) reported that, for maximum stability of most analytes,
DBSs should be stored at low temperatures and controlled low humidity.
We conducted the thermal stability evaluations reported here to test
the stability of the AARM materials that were maintained in a dark
environment at <30% humidity while stored at temperatures that might
be encountered in transit (increased and ambient temperatures), in
routine storage for daily laboratory use (refrigerated at 4 °C), and
in long-term frozen storage (-20 °C). Only after prolonged exposure
to increased temperature (37 °C) did the AARM materials show
significant loss of amino acid concentrations. Using regression slopes
and y-intercepts from analyses of the 37 °C storage data,
we estimated that Phe, Leu, Tyr, and Val would retain 83–85% of their
initial concentrations after storage for 30 days at 37 °C, whereas
Met would retain ~76% of its initial concentration after 30 days at
37 °C. Our observation that Met is less stable in storage than Phe,
Leu, and Tyr is consistent with previously reported studies on the
stabilities of newborn screening blood-spot samples stored at room
temperature for periods 
2 years (17). Because the
P values derived from analyses of samples stored at ambient
room temperatures and under refrigeration were >0.05, we concluded
that no statistically significant loss of amino acid concentration
occurred during the time intervals tested. We detected no loss of
analyte concentration from the materials stored for 1 year in the dark
at -20 °C and <30% humidity; longer-term evaluation of the
stability of the AARMs at -20 °C is in progress.
Only two sources of filter paper have been approved by the Food and Drug Administration for blood collection in the United States. These papers, Grade 903 (Schleicher & Schuell) and BFC 180 (Whatman), must meet the national criteria defined for acceptable performance (18). Because performance can differ among filter papers (19)(20), DBS reference materials for use in the US must be prepared on an approved paper source. Hematocrits and the size of the blood spot are also important performance variables, and they should simulate the routine specimen being collected from the newborns. Each of these variables affects the retained serum volume in an aliquoted punch from the blood spot. The Phe DBS reference material produced in Europe (15) was not prepared on a US-approved paper source, and the blood-spot volumes are much smaller (50% or more) than those routinely used in the US for newborn screening. The amount of serum in a given paper-punch-sized blood aliquot increases with the volume of blood applied to the paper, but this increase may not be directly proportional to the volume applied. The exact nature of this relationship is difficult to predict and may be influenced by several variables, such as red blood cell lysis, room humidity, force of application, point or style of application (chromatographic effects), and type of filter paper. Our multi-amino acid DBS materials were prepared under monitored environmental conditions and on US-approved paper with precise blood volumes simulating the collected volume for screening laboratories in the US and several other national screening programs.
Most screening programs analyze for several amino acids in addition to Phe. Furthermore, the multi-amino acid calibrator allows laboratories to use amino acid ratio comparisons (21) for improved quality-assurance efforts. Because mothers and babies are being discharged from birthing centers earlier, screening programs need more sensitive and specific methods of testing to reduce testing redundancy and improve follow-up operations for babies with metabolic disorders. These changes in newborn screening have driven the improvement and shift in testing technologies for laboratories. The new technologies need high accuracy-based calibration materials for multiple amino acids in a single calibration material to complement the testing technologies (e.g., HPLC and MS/MS).
In an effort to demonstrate and quantify the impact that standardized calibration of DBS methods has on the measurement of amino acids, we will distribute sets of AARMs, along with a panel of test samples, to a group of manufacturers and screening laboratories that use different analytical methods. This standardization process initiated by the availability of common reference materials will focus efforts that can lead to enhanced overall interlaboratory comparability. In Europe, the recently released Phe reference material has reportedly demonstrated improved interlaboratory comparability (15), and similar improvements should occur for the five amino acids with the use of the AARMs in US newborn screening laboratories and other laboratories using the same type of blood-collection paper and larger blood-spot collections.
These candidate DBS reference materials for method calibration will be primarily directed toward producers of screening-test products; however, requests from screening laboratories will be considered on the basis of their justified need. Initially, the distribution will not be very restrictive because of the large quantity of materials available (1300 sets) and because several screening laboratories use noncommercial methods. Notification of the availability of materials will be made by an announcement in appropriate newsletters. The AARMs will be stored at CDC and distributed from CDC. On the basis of the production quantity of these materials and the documented stability of the amino acids, we anticipate the AARMs to be available for at least 5 years, especially because they are kept in continuous storage under optimal conditions. We will monitor the stability of the materials during the storage intervals and take appropriate actions to produce new materials when a significant change in analyte concentration is observed.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. W. Adam, J. R. Alexander, S. J. Smith, D. H. Chace, J. G. Loeber, L. H. Elvers, and W. H. Hannon Recoveries of Phenylalanine from Two Sets of Dried-Blood-Spot Reference Materials: Prediction from Hematocrit, Spot Volume, and Paper Matrix Clin. Chem., January 1, 2000; 46(1): 126 - 128. [Full Text] [PDF]
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