Using Tandem Mass Spectrometry for Metabolic Disease Screening Among Newborns
A Report of a Work Group
The following CDC staff members prepared this report:
W. Harry Hannon, Ph.D.
Scott D. Grosse, Ph.D.
National Center for Environmental Health
in collaboration with Bradford L. Therrell, Jr., Ph.D.
National Newborn Screening and Genetics Resource Center
William J. Becker, D.O.
State Department of Health
Donald H. Chace, Ph.D., M.S.F.S.
Neo Gen Screening, Inc.
George C. Cunningham, M.D.
California Department of Health Services
George F. Grady, M.D.
University of Massachusetts Medical School
Gary L. Hoffman
Wisconsin State Laboratory of Hygiene
Marie Y. Mann, M.D., M.P.H.
Health Resources and Services Administration
Joseph Muenzer, M.D., Ph.D.
University of North Carolina
Chapel Hill, North Carolina
John J. Mulvihill, M.D.
University of Oklahoma
Oklahoma City, Oklahoma
Susan R. Panny, M.D.
Maryland Department of Health and Mental Hygiene
"Public health agencies (federal and state), in partnership
with health professionals and consumers, should continue to develop
and evaluate innovative testing technologies [and] design and apply
minimum standards for newborn screening activities . . . ."
Newborn Screening Task Force, May 1999
Increasingly, tandem mass spectrometry (MS/MS) is being used for
newborn screening because this laboratory testing technology substantially increases
the number of metabolic disorders that can be detected from dried
blood-spot specimens. In June 2000, the National Newborn Screening and
Genetics Resource Center, in collaboration with CDC and the Health Resources
and Services Administration, convened a workshop in San Antonio, Texas.
Workshop participants examined programmatic concerns for health providers choosing
to integrate MS/MS technology into their newborn screening activities.
Representatives from approximately 50 public and private health agencies and
universities participated in the workshop. The workshop participants and work group
focused on laboratory methodology, decision criteria, quality assurance,
diagnostic protocols, patient case management, and program evaluation for using MS/MS
to analyze dried blood spots routinely collected from newborns. This work
group report contains proposals for planning, operating, and evaluating
MS/MS technology in newborn screening and maternal and child health programs. As
a supplement to these proposals, this report contains synopses of
selected presentations made at the 2000 workshop regarding integration of
MS/MS technology into newborn screening programs. The proposals contained in
this report should assist policymakers, program managers, and laboratorians
in making informed decisions regarding the process of including MS/MS
technology in their newborn screening and maternal and child health programs.
Each year, approximately 4 million babies in the United States have dried blood
spots analyzed through newborn* screening programs. This screening is intended to
detect inborn disorders that can result in early mortality or lifelong disability. Detectable
disorders include metabolic disorders (e.g., phenylketonuria [PKU]), hematologic
disorders (e.g., sickle cell disease), and endocrinopathies (e.g., congenital hypothyroidism).
three groups of disorders account for approximately 3,000 new cases of potentially
fatal or debilitating disease each year for which outcomes are improved with early
identification and treatment through newborn screening systems. The introduction of
tandem mass spectrometry (MS/MS) in the 1990s for population-based newborn screening
has enabled health-care providers to detect an increased number of metabolic disorders in
a single process by using dried blood-spot specimens routinely collected from
newborns (13). However, using MS/MS in newborn screening programs is new, and scientific
data are limited regarding incorporating this technology into newborn screening and
maternal and child health programs.
MS/MS technology enables improvements in and consolidation of metabolic
screening methods to detect amino acid disorders (e.g., PKU, maple syrup urine disease,
and homocystinuria) among newborns, and does so with a low false-positive rate
(46). MS/MS technology expands the metabolic disorder screening panel (i.e., the number
of disorders that can be detected) by incorporating an acylcarnitine profile, which
enables detection of fatty acid oxidation disorders (e.g., medium-chain acyl-CoA
dehydrogenase [MCAD] deficiency) (7-10) and other organic acid disorders. MS/MS can reliably
analyze approximately 20 metabolites in one short-duration run (i.e., ~2 minutes) and provide
a comprehensive assessment from a single blood-spot specimen (Table 1).
Screening for multiple disorders in a single analytical run by using MS/MS
requires that program administrators and laboratorians choose which types of conditions are
to be screened. For example, laboratory A uses MS/MS to detect amino acids only;
laboratory B uses MS/MS to detect acylcarnitines only; and laboratory C screens for both.
In addition, other technical concerns must be addressed before MS/MS technology can
be integrated effectively into a newborn screening program, including deciding
which analytes to use in characterizing each disorder (e.g., octanoylcarnitine analysis can
indicate both MCAD deficiency and multiple acyl-CoA dehydrogenase deficiency).
Studies are limited regarding use of MS/MS technology in newborn screening
programs, but existing studies indicate that screening a full panel of acylcarnitines
and amino acids yields rates of 1:4,0001:5,000 for MS/MS-detectable disorders
(16-13). For certain metabolic disorders, early detection can result in substantial improvements
in health outcomes. For example, MCAD, which has an incidence rate of
1:10,0001:20,000 newborns, results in substantial morbidity and reported mortality rates of
20%25% among infants and children during the first 3 years of life
(14). Effective treatment is available, and detection and intervention before onset of illness can prevent
mortality and improve the quality of life for MCAD patients. Although effective treatments do
not exist yet for certain other metabolic disorders identifiable by MS/MS testing
(1), patient and family advantages can still be achieved through early diagnosis
(15). Also, MS/MS can detect metabolic disorders after an illness occurs, even if that illness occurs after
the newborn period (Table 2).
June 2000 Workshop and Work Group Goals
In June 2000, the National Newborn Screening and Genetics Resource Center,
in collaboration with CDC and the Health Resources and Services Administration
(HRSA), convened a workshop in San Antonio, Texas, to examine programmatic concerns
for effectively integrating MS/MS technology into newborn screening programs.
Representatives from approximately 50 public and private health agencies and universities
participated in the workshop.** The participants focused on laboratory methodology,
decision criteria, quality assurance, diagnostic protocols, patient case management,
and program evaluation. This report contains their proposals for planning, operating,
and evaluating MS/MS technology in newborn screening and maternal and child health
programs. Their proposals are included in this report to assist policymakers, program
managers, and laboratorians in planning state-mandated screening programs or
optional metabolic testing through partnering of state and private screening laboratories.
The workshop participants did not address newborn disorders that are screened by
other technologies and that should be considered for a comprehensive newborn
screening panel (e.g., sickle cell disease, congenital adrenal hyperplasia, galactosemia,
biotinidase deficiency, and cystic fibrosis). Therefore, these proposals address MS/MS
technology only. Further, as a supplement to their proposals, this report contains synopses of
selected presentations made at the 2000 workshop regarding integration of MS/MS
technology into newborn screening programs (Appendix).
Specifically, workshop participants focused on the following concerns
for policymakers, program managers, and laboratorians interested in MS/MS testing
for newborn screening programs:
What barriers exist that impede MS/MS implementation?
What technical concerns exist regarding instrumentation (e.g., throughput,
cost, software, accessories, or capabilities)?
What are the approaches and cutoff decisions for identifying
presumptive positive test results?
What guidance do program managers and laboratorians need who are
planning to use, are using already, or are evaluating MS/MS technology for
What are the expectations and resource needs for follow-up,
diagnostic confirmation, parental genetic counseling, and patient case management?
What are the medical concerns for parents and health-care providers,
including identification and treatment of MS/MS-diagnosed disorders?
How would program evaluation be conducted for MS/MS integration,
including quality control and proficiency testing?
In their discussions, workshop participants considered the role parent support
groups*** and advocacy organizations are taking in promoting inclusion of MS/MS technology
in newborn screening services. This increased advocacy has resulted in increased
media attention regarding the health burden of metabolic disorders among newborns
(16,17). Consequently, more newborn screening program administrators and maternal and
child health-care providers are considering integrating this technology into their
programs (18). Key factors in deciding to implement MS/MS are its versatility and ability to
detect additional treatable metabolic diseases, including fatty acid and organic acid
oxidation disorders. However, medical literature is limited regarding use of MS/MS technology
in newborn screening programs. Furthermore, the identification of metabolic
disorders must be confirmed by validated scientific methodologies. Additional studies are
needed to assess effectiveness of MS/MS, and sustained discussions among those persons
involved in using or contemplating using MS/MS should be ongoing to enable
policymakers, program managers, and laboratorians to make more informed decisions.
In the 1930s, amino acid metabolism disorders and in the 1970s, fatty acid
oxidation disorders were recognized as causes of morbidity and mortality among infants
and children. As metabolic disorders were recognized, researchers worked to develop
methods to detect them. In the 1960s, population screening for amino acid disorders
began, and in the mid-1980s, one researcher demonstrated the therapeutic value of
measuring the fatty acids released from acylcarnitines
(19). Early analytical methods required hydrolysis of fatty acids from acylcarnitines or analysis of urinary organic acid profiles
as primary analytical approaches to diagnosing inborn errors of metabolism
(20,21). Acylcarnitines are quaternary ammonium salts that are not readily analyzed by
gas chromatography/mass spectrometry, a readily available clinical chemistry method.
With the introduction of fast-atombombardment ionization techniques for MS, ionic
compounds with high polarity (e.g., acylcarnitines) could be analyzed with simple
preparation techniques (22). Use of MS/MS for acylcarnitine analytes in plasma eliminated
time-consuming chromatography but maintained method specificity
(23,24). Rapid analyses of highly polar compounds made possible use of MS/MS in newborn screening,
which requires rapid high throughput to be cost-effective. Also, during the 1980s,
improved diagnostic skills enabled better recognition of disorders of intermediary metabolism.
In 1990, analyses of amino acids present in dried blood-spot specimens were used to
document newborn screening applications of MS/MS
(4,5,10,25). Existing methodology for the MS/MS analyses of acylcarnitines was modified and combined with that for
amino acids in an approach that remains relatively unchanged.
During the early 1990s, improvements were made in automated analysis, in
part enabled by the introduction of electrospray ionization, sample-introduction
techniques, method validation, and development of automated interpretation systems
(8,9,11,12,2629). Concurrently, the number of specimens analyzed annually increased
substantially. From the first analyses in 1990 of 510 specimens/day in clinical testing laboratories
to early pilot studies in 1993 of 60120 specimens/day
(11), the use of MS/MS technology has grown exponentially so that, during 2000, an estimated 500,000
were analyzed (Figure 1).
With increased demand for expanded newborn screening, MS/MS technology
has been successfully implemented in private sector and public health laboratories
across the United States (Figure 2). Routine, state-sponsored screening using MS/MS is
performed in the District of Columbia, New England (i.e., Massachusetts, New
Hampshire, Vermont, Maine, Rhode Island), North Carolina, and Wisconsin. However, the
technology is used for screening acylcarnitine profiles in only three of these states:
Massachusetts, North Carolina, and Wisconsin. In addition, optional supplemental testing (i.e., testing
for diseases not included in the selected panel of a state screening program) is offered for
a fee by Neo Gen Screening, Inc.***** (Bridgeville, Pennsylvania) and the Institute of
Metabolic Disease (Baylor University Medical Center, Dallas, Texas) to parents, physicians,
and hospitals either directly or through state newborn screening programs. Pilot testing
is under way in Illinois, Iowa, Louisiana, and Ohio; and California, Minnesota, and New
York have purchased MS/MS equipment.
Policymakers and program managers need to be familiar with standards for
MS/MS testing when soliciting, contracting, or evaluating newborn screening laboratory
services. Work group proposals regarding implementation and operation of MS/MS
laboratories are provided here with the understanding that this technology is new and,
therefore, changing rapidly. Although modifications throughout the testing system are to be
expected, all changes in laboratory practice should adhere to published laboratory
guidelines before being implemented.
Basic knowledge of MS/MS technology standards and laboratory practice
requires an understanding of the scientific terminology.
Work Group Proposals
The technology should be referred to as
tandem mass spectrometry, not tandem mass
The standard abbreviation for tandem mass spectrometry is
MS/MS, not TMS or TM/TM.
MS/MS represents two mass spectrometers joined by a
A universal description of the analytes and disease states is needed. For
example, succinate and
methylmalonate can refer to the same analyte.
MCAD and MCADD are used interchangeably to refer to medium-chain
acyl-CoA dehydrogenase deficiency. A task force of laboratorians and clinical
specialists should work to resolve such nomenclature differences.
Acylcarnitine results should be reported in micromolar (µM) units (whole
blood), and amino acids should be reported as micromolar (µM) (whole blood) with
the concentration in milligrams per deciliter (mg/dL) given in parentheses.
Standard and Sample Preparation
Work Group Proposals
Sample preparation techniques for acylcarnitines and amino acids should
be validated in accordance with the Clinical Laboratory Improvement
Amendments of 1988 (30) and good laboratory****** and
measurement practices (31).
All reagents, buffers, and solvents should be high-pressure liquid
chromatography grade or better.
Validated methods should be published in peer-reviewed journals.
When stable-isotope internal-standard methodology is used, the labeled
internal standard should be identical to the analyte of interest. If the analyte is
not available in the labeled form, the nearest homologue can be substituted.
For example, the diagnostic analyte is phenylalanine for PKU, which requires
phenylalanine, or equivalent, for quantitation. However, not all acylcarnitines
will be available as internal standards for detecting fatty acid oxidation or organic
acid oxidation disorders by measuring acylcarnitines levels. Therefore, for a
complete acylcarnitine profile, D9-C0,
D3-C3, D3-C4, D9-C5,
D3-C14, and D3-C16 should be used as internal standards. Other isotopes can be used if they are
properly validated. Limited profiling might involve fewer internal standards but
still requires validation.
Individual and premixed stable-isotope internal standards are
commercially available. Commercial suppliers should include a statement regarding
the standard material's concentration and stability. Dilutions of commercial
standards or in-house preparations should be validated by analyzing unlabeled
materials (i.e., controls) before use.
Stability of internal-standard or quality-control preparations should be
validated and documented for each laboratory performing MS/MS analyses.
Testing protocols should specify what safety recommendations,
universal precautions, personal protective gear, and environmental controls are required.
Sample preparation areas should be physically separated from instrument
areas to avoid contamination. One MS/MS instrument should have a capacity of
500 samples/day. Good laboratory and measurement practices should be
Instrument Resources and Calibration
Work Group Proposals
Manufacturer's guidelines for power requirements, exhaust
specifications, laboratory gas purity and pressure, and laboratory environment should
be followed. All MS/MS used for metabolic disease detection requires
high-purity compressed nitrogen delivered at a specified pressure. This gas can be
supplied by nitrogen generators, compressed gas tanks, or Dewar tanks.
Certain instruments require uninterrupted gas flow; therefore, the ability to change
tanks regularly without interruption of flow or loss of pressure is required (i.e.,
empty tanks connected to the system must be replaced with filled tanks before all
tanks become empty).
Additional peripheral equipment for MS/MS is required, including a
liquid chromatography pump, syringe pump for calibrating and tuning, and
an autosampler (preferably one capable of holding multiple 96-well
microplates). Sample preparation equipment might include sample concentrators, forced
air ovens or equivalent, and reagent-dispensing devices (either automated or
hand-held). Nearby telephone access is desirable for instrument troubleshooting.
Operators of MS/MS instruments should hold a minimum of a bachelor of
science degree in a laboratory science or medical technology. In addition, they must
meet the pertinent Clinical Laboratory Improvement Amendments of 1988
requirements (30). Additionally, MS/MS laboratorians should have a)
mechanical aptitude, b) computer skills, and c) an interest in mass spectrometry
technology. Each instrument requires one primary laboratorian and a backup.
Laboratories having multiple instruments should have an equal number of personnel plus
one or two laboratorians, depending on whether the supervisor serves as the backup.
Managers and supervisors of MS/MS operations should have
background experience in mass spectrometry. One manager is sufficient to oversee
Newborn screening laboratories should develop a backup plan for
instrument downtime. That plan should include ready access to additional instruments
or backup laboratories.
Each instrument manufacturer's recommended calibration procedure should
be followed. These procedures involve using a standard material (e.g.,
sodium iodide, rubidium iodide, polypropylene glycol, or similar compound).
Mass scales should be calibrated above and below the mass range of
interest. Calibration from 23 megahertz to 600 megahertz should suffice for analyses
being performed in metabolic disease investigations.
Users should prepare an instrument check solution at a defined
concentration comparable to patient specimens in mass and intensity. This solution should
be used daily to measure the sensitivity of the instrument. Results, as counts
or signal intensities, should be recorded each day and a minimum signal
intensity established as an alert of a possible change in instrument performance.
Check solutions should contain the same mix of standards used to calibrate the assay.
A higher concentration tuning solution should be available also and separate
from the routine check solution. This tuning solution enables optimization
of parameters for individual acylcarnitines that might have variable
ionization efficiencies of short- versus long-chain acylcarnitines and neutral versus
basic amino acids.
Reducing Instrument-to-Instrument Variability
Work Group Proposals
Because quantitative results for raw ion counts can vary from instrument
to instrument, a minimum sensitivity threshold for all instruments should be
defined before use. Concentration calculations using ion ratios (i.e., the mass of
unlabeled analyte versus labeled internal standards) should vary
<10% from instrument to instrument. NCCLS******* voluntary consensus definitions of quantitation
and detection limits should be used (32).
When the concentration of an analyte is close to the detection limits of the
method, as for certain acylcarnitines, differences between apparent normal results
from one instrument to another can result from electronic signal noise. This
should decrease as a particular analyte level increases and should be minimal
in true disease states when multiple instruments are properly maintained.
Quality Control, Proficiency Testing, and Quality Assurance
Work Group Proposals
Quality control should consist of
>2 control specimens/96-well microplate.
One control should contain analyte concentrations above the abnormal
reporting level, and the second control should be at or near the abnormal cutoff value.
The fatty acid oxidation disorder and organic aciduria disorder controls should
contain as many of the acylcarnitines as are commercially available.
A reagent blank should be included on each plate and should be located after
the high-level control to monitor analytical carry-over.
The daily patient mean, or median, for each analyte should be monitored to
follow method performance.
A schedule for routine maintenance should be established for
quality performance. Maintenance schedules should follow manufacturers'
recommendations and each instrument's operational experience.
External quality-control specimens should be analyzed periodically.
CDC's Newborn Screening Quality Assurance Program, operated in collaboration
with the Association of Public Health Laboratories, can provide control materials
for amino acids and acylcarnitines.******* For acylcarnitines, the calculated
concentrations are dependent on the internal standard to which the unlabeled acylcarnitine
is compared. Therefore, when interlaboratory comparisons are performed,
the internal standards used to calculate the reported values need to be defined.
Using a relative response factor by dividing each laboratory's observed value by
the expected value might be advantageous when interlaboratory performances
A government-supported external proficiency testing program is needed
for newborn screening laboratories using MS/MS testing
(1). CDC's Newborn Screening Quality Assurance Program is pursuing addition of a dried
blood-spot proficiency testing program for MS/MS-detectable disorders. Proficiency
testing challenges should include quantitation of analytes and assessment of
the laboratory's capability to recognize disease profiles. CDC's program is
producing dried blood-spot materials for amino acids and acylcarnitines quality control
and proficiency testing purposes, but problems remain with standardization of
such analytes as glutarylcarnitine and hydroxyacylcarnitines for which no
synthetic material is available. When external proficiency testing is not available,
an interlaboratory specimen exchange program with documented results should
be established to assess accuracy in detecting metabolic disease profiles.
Results of a newborn screening test might be affected by the patient's
medical treatment before specimen collection.
Research is needed regarding the effect
of carnitine-fortified total parenteral nutrition and hyperalimentation
solutions, transfusions, prescription drugs, and metabolites that can affect the MS/MS
test results. Discrepancies among multiple sample collections should be
discussed with metabolic specialists, physicians, and the patient's primary care provider.
MS/MS, although specific and accurate, is one of multiple tests performed
in newborn screening laboratories. As with other tests, abnormal results should
be confirmed by additional diagnostic testing, including MS/MS serum analysis,
gas chromatography/mass spectrometry urinary organic acid analysis, or
quantitative high-pressure liquid chromatography using ion exchange or
similar approaches, depending on disease confirmation.
An electronic communication system (e.g., an on-line bulletin board
service) should be used for information exchange among laboratories regarding
MS/MS operational issues, problems, and
The Association of Public Health Laboratories, in collaboration with
other organizations, should sponsor a regularly held workshop (e.g., annually)
for newborn screening MS/MS instrument users. In addition, a session regarding
MS/MS should be included at national and regional conferences on
Interpreting MS/MS Data and Reporting Results
Work Group Proposals
Cutoff values for reporting abnormal levels for each analyte should
be established by using statistical measurements (e.g., percentiles, means,
and standard deviations) in consultation with metabolic disease specialists. A
multitier system could be designed, depending on methodology, metabolic disorder,
and target population. Cutoff values should be compared with published ranges
but should be individualized to the methodology used and patient population.
Cutoff values should be adjusted up or down, on the basis of periodic re-evaluations
or changes in methodology or population distribution. Unless
interinstrument performance comparability is
<10% (i.e., concentration calculations),
instrument-specific cutoff values should be developed. Programs with multiple
instruments should be able to perform satisfactorily for a single cutoff value.
Laboratories can elect to establish two levels of abnormal results. One
level would be the concentration that is indicative of a particular disorder. Test
results greater than that concentration would require immediate referral to the
clinical management team for follow-up. The second level would be a
borderline concentration that would require resampling and retesting. Decisions to use
this two-level system might be dependent on the availability of follow-up resources.
In addition to defining analyte concentrations or to profiling analytes, a ratio
of different analytes might be helpful in data interpretation. For example,
the phenylalanine/tyrosine ratios and C8/C10 acylcarnitine ratios might reduce
false-negatives and false-positives in PKU and MCAD detection, respectively.
In addition to establishing a high cutoff value, setting a low cutoff value for
certain analytes would be useful. For example, a low, free carnitine might mask
the presence of a disorder (i.e., if low or undetectable carnitines are
present, acylcarnitines cannot be formed).
For specimens collected from newborns aged >1 week, the
acylcarnitine measurements should be examined closely because acylcarnitines
levels decrease significantly with age. Establishing abnormal cutoffs for older babies
is an option, but could be difficult. Timing of specimen collection might be critical
in selected cases and result in invalid second specimens. Repeat testing of
second specimens collected from older newborns should not be considered a
reliable specimen for confirmation of all disorders.
Reporting MS/MS results is the responsibility of each newborn
screening program. Screening laboratories should employ a trained, credentialed person
to interpret screening profiles, similar to requirements for MS/MS
diagnostic laboratories. What results to report and how to report them should be decided
in consultation with state-designated referral centers, health-care practitioners,
and public health follow-up staff. Options include
--- providing test data for both normal and abnormal results with or
--- providing only interpretation (i.e., test results are normal or abnormal); or
--- combining these two options and reporting normal results as an
interpretation only (e.g., fatty acid oxidation is normal) and reporting abnormal results
as observed values with interpretations.
Program managers should be aware that no U.S. Food and Drug
Administration-approved interpretive software for MS/MS newborn screening exists.
Thus, individual laboratory validation of interpretive protocols is required.
Specimen and Control Sample Storage
Work Group Proposals
Stored patient specimens and control samples must be kept frozen at
<20 C with humidity <30% to ensure long-term storage validity. Desiccants should be used
to maintain humidity levels. Laboratories should maintain written policies
and procedures that specify storage standards for specimens
When patients' specimens are analyzed after storage, control samples
stored under the same conditions should also be used. Results of analyzing
stored specimens should be interpreted cautiously because long-term validation
studies have not been published.
NEWBORN SCREENING FOLLOW-UP
Workshop discussions regarding patient follow-up were consistent with those of
the Newborn Screening Task Force meeting, which was convened by the American
Academy of Pediatrics in Washington, D.C. in 1999
(1); therefore, proposals regarding follow-up concerns are limited in this report. Newborn screening follow-up includes short-
and long-term components. Short-term follow-up tracks patients from a positive test
result through diagnosis and acknowledgment by a health-care provider of that
diagnosis. Long-term follow-up tracks patients from diagnosis through clinical management
and beyond to ensure that they and their family members receive needed services.
Work Group Proposals
The same short-term follow-up approach should be followed for
MS/MS-detectable disorders as for conditions in which delayed treatment poses a
high risk of fatal outcomes and for which timely transport of test samples and
analysis are essential (e.g., galactosemia and congenital adrenal hyperplasia).
For metabolic disorders in which usual feeding practices might result in acute
crisis and risk of death, upon notification of an initial abnormal test result,
physicians should advise parents of what short-term feeding measures to take in advance
of repeat and confirmatory tests so as to avert a potentially lethal crisis
(33). In the case of MCAD deficiency, an infant who experiences fasting as the result of loss
of appetite is at risk of hypoglycemic crisis, which is preventable if parents
ensure that children eat regularly (34).
Although reported false-positive rates are low and metabolic disorders are
rare, an increase in the number of diagnosed disorders will require additional
follow-up personnel and definitive diagnostic services. Newborn screening
program personnel will need technical and resource assistance with
developing educational materials and in training staff and health-care providers in
follow-up procedures for MS/MS-detectable conditions.
To conduct adequate long-term follow-up, programs will need to establish
or improve patient-tracking systems. Ideally, data management for such a
system would include registries to which treatment centers continually provide
updated information, treatment compliance, and outcomes.
DIAGNOSIS AND TREATMENT
MS/MS technology can assist in diagnosing metabolic disorders during the
newborn period that previously were diagnosed only after symptoms developed.
Presymptomatic detection now allows treatment initiation while the infant is healthy and assists in
defining the spectrum of clinical disease related to these disorders. MS/MS technology can
be used advantageously to screen for selected amino acid disorders for which other
newborn screening methods are used. For example, MS/MS can accurately detect
elevated phenylalanine levels in dried blood spots taken from infants as young as age
<24 hours. By using the MS/MS-detected phenylalanine/tyrosine ratios, physicians can
diagnose PKU earlier and rely on an assay with a reduced false-positive rate
For policymakers and program managers, the uncertainty of outcomes from
early diagnosis complicates deciding whether to use MS/MS for newborn screening. For
example, presymptomatic MCAD-deficiency detection might lead to decreased
morbidity and mortality among infants, whereas evidence regarding outcomes for other fatty
acid oxidation disorders is lacking (35). Because data are still limited, additional research
is needed to determine what interventions work for MS/MS-detectable metabolic
disorders. Consensus is lacking regarding which disorders should be included in a
MS/MS screening panel and for defining a list of mandated tests in the panel. Additional
pilot testing is required. Another challenge to the definitive diagnosis of metabolic disease
is that persons with one of these disorders might by affected in different degrees of
severity (i.e., clinical heterogeneity) with varying physical and biochemical
manifestations. Opinions differ as to where to draw the line on the diagnosis of infants as affected or
not by a particular disorder. In using MS/MS technology, clinical heterogeneity
presents challenges in setting cutoffs to minimize the frequency of false-positive and to
prevent false-negative results. The full clinical spectrum of metabolic disorders is unknown
because certain MS/MS-detectable disorders are rare and not well-described in the
literature. Further, even for classical cases, different mutations in DNA (deoxyribonucleic
acid) can exist. Milder variants exist also, and their natural history is unclear. The term
milder variant is based on the discovery of a mutation that does not correlate with
clinical symptoms and is recognized only when a particular stress is placed on the affected
child. Newborns with mild or late-onset variants of metabolic disorders are more likely to
be missed by MS/MS. Conclusions based on the outcomes of limited numbers of
reported cases are not valid assessments of variants; therefore, prevalence data for variant
cases will be sparse until statistically significant numbers of test results are collected
In conjunction with diagnostic questions needing additional research, treatment
outcomes are of concern for policymakers and program managers considering MS/MS
for newborn screening. Again, long-term studies are needed to evaluate whether
outcomes are improved as a result of MS/MS screening and early diagnosis. Clinical treatment
and long-term care services are costly; therefore, treatment expense and funding
resources for support services are of concern.
Work Group Proposals
To take advantage of MS/MS technology for detecting abnormal metabolite
levels among newborns, many of which are present during the first week of life,
rapid transport of blood-spot specimens to the screening laboratory is required.
The time from birth to diagnosis should be as short as possible, with an ideal
time frame of <5 days.
Efforts should be made to reduce handling time within hospitals to decrease
time to analysis.
Optimal specimen transport time from the hospital to the laboratory is
<24 hours, but transport time of
<48 hours is critical. Program managers should
consider using courier services and requiring 24-hour delivery of specimens to
the laboratory. To ensure delivery, program managers should have
contingency plans in place.
A 6-day work schedule for MS/MS laboratories performing newborn screening
is preferable because the first analysis should be performed within a
24-hour turnaround time, with another <24 hours for retesting and confirmation if
test results indicate abnormal levels.
For all infants suspected of having a metabolic disorder, confirmatory
testing, using published standard metabolic testing procedures, should be
performed before treatment. Different criteria should be used for diagnostic
confirmation testing, but MS/MS technology can be used also.
Acylcarnitine analysis performed by an MS/MS laboratory is valid for
specimens from newborns but might not be for specimens from older infants. Duplicating
an analysis by the same or another laboratory adds only limited information, and
the results could be misleading. Laboratory results should be correlated with
the clinical status. If available, a DNA analysis for common mutations in the
disorder would provide further confirmation of the disease.
Treatment resources might be inadequate as a result of the rarity of
metabolic diseases among newborns; therefore, ensuring delivery of clinical services
can be a logistical and funding challenge for health-care providers. Access
to treatment services that includes specialized care centers, nutritionists,
social workers, certified genetics counselors, and specially prepared medically
required foods must be ensured before screening is introduced.
Because of the emotional and financial burden that care, treatment,
and outcomes for newborns with MS/MS-detectable metabolic conditions pose on
the families, clinicians should communicate concerns and treatment options
carefully to parents of possibly affected children
MS/MS SCREENING EVALUATION
The Newborn Screening Task Force recommends that state and territorial
include evaluation, performance monitoring, and quality assurance activities
in their newborn screening systems;
conduct oversight of program operations; and
monitor and evaluate program performance through collection,
assembly, analysis, and reporting of data, including outcome evaluations
Specifically, the evaluation of a screening program involves examining the
clinical validity (i.e., sensitivity and specificity, positive or negative predictive values),
clinical utility (i.e., improvement in health or development outcomes with early treatment),
and cost-effectiveness of the screening protocol for each disorder
(36). Pilot demonstration programs in states could provide information regarding certain variables if they
had adequate resources to acquire and report technical and clinical results
(15). Determination of false-positives, specificity, and positive predictive value is straightforward and
can be calculated using a data system that tracks infants from initial test results
through diagnosis. Remaining variables require development of more sophisticated and
rative data collection systems, particularly for evaluating the clinical utility of
screening, which for any given disorder, depends on the demonstration that early treatment
improves long-term outcomes.
Work Group Proposals
Long-term storage of leftover specimens is a critical consideration for
newborn screening programs. Leftover specimen storage and use should be guided
by policies and procedures that include protection against their inappropriate use
(1). Retention of blood-spot specimens could be pivotal in determining
false-negative rates (1). False-negatives can be confirmed only by identification of an
affected patient clinically or through autopsy findings, and comparing those findings
with results obtained by retesting the original blood spot and using
storage-control specimens. Correct storage of specimens is required for this process
A key challenge to using MS/MS for expanded newborn screening is the lack
of published scientific data regarding MS/MS-detectable disorders
among newborns. Specifically, data are needed regarding the results of diagnosis
and treatment to justify the expanded screening. Expert opinion regarding
the justification for performing expanded screening varies substantially
(1). A list of the disorders detectable by MS/MS are provided in this report (Table 1).
Expert reviewers have concluded that MCAD, one of the disorders that requires
MS/MS screening, meets almost all of the criteria to justify newborn screening, and
these reviewers recommend collecting additional data through pilot MS/MS
screening programs (35,37,38). MS/MS also offers certain advantages over
traditional methods for detection of PKU and other amino acid disorders
Although certain newborn screening programs are expanding without
scientific support, program managers should incorporate epidemiologic research
methods into implementation efforts so that evaluation results can be used by others
facing this challenge.
To assess the utility of expanded MS/MS screening, national data
regarding screening performance and outcomes should be collected. No
mechanism beyond the National Newborn Screening and Genetics Resource Center's
report is in place for collection of these data. Federal agencies could support the
design and implementation of projects targeted for gathering data and
retrospectively analyzing the experience of expanded newborn screening programs, but
such projects first require development of uniform data reporting protocols.
In particular, such projects would require agreement regarding consistent
case definitions, including normalization of cutoffs.
Routine data collection by a single state program is unlikely to be sufficient
to evaluate the long-term outcomes of screening for these conditions.
Constructing prospective cohorts of patients with rare disorders, although expensive, is
one way to address issues of the true incidence and prognosis of these
disorders. Prospective cohorts have been constructed for other diseases, notably
childhood cancers and hemophilia. Another relevant model for expanded
screening research is the multicenter registry of cystic fibrosis patients coordinated
supported by the Cystic Fibrosis Foundation. A federally funded multicenter
study could track newborns with positive test results and actively pursue other
clinically detected cases. This study would require a substantial long-term
funding commitment and would require the expertise and cooperation of
epidemiologists and health services researchers in multiple federal agencies.
Collection of economic data regarding costs and cost savings is essential
for analyzing the cost-effectiveness of screening. Collecting economic data
requires improved coding techniques for inborn errors of metabolism so that use of
health-care services is consistently recorded. Consistent recording would allow
financial data collection to justify continuation of programs or
An epidemiologic perspective should bring additional benefits (e.g., definitions
of minimal essential data and improved data coding). Other strategies
besides prospective cohort methods are possible. For example, data from
medical examiners in different states has led to the realization that some sudden
and unexplained childhood deaths can be attributed to specific inborn errors
of metabolism with a higher frequency than previously recognized.
Public health officials and newborn screening program managers
evaluate screening systems differently than parents of affected children,
primary-care providers, and the public do. Public health officials and program managers
focus on positive and negative predictive values and clinical utility. In contrast,
the public and physicians without substantial experience with these disorders
lack understanding regarding screening and might have unrealistic expectations
for treatment outcomes. Both parents and health professionals need to be
educated regarding limitations and availability of expanded newborn screening
The goals of the 2000 workshop were to provide guidance for newborn
screening program managers and policymakers who are using or planning to use MS/MS
technology; workshop goals did not include recommending screening for specific
MS/MS-detectable disorders. Interest in using MS/MS technology for newborn screening for
an expanded range of inheritable metabolic disorders is increasing throughout the
United States. A limited number of public health screening laboratories have introduced
MS/MS with minimal difficulty. Others have started MS/MS testing with a limited
understanding of MS/MS applications and detectable disorders; those programs have found
installation and use of MS/MS instrumentation to be a substantial undertaking. In certain
cases, overall system concerns have not received adequate consideration
(1). The American College of Medical Genetics and the American Society of Human Genetics state that ".
. . MS/MS can provide substantial benefit to patients and their families, if
thoughtfully integrated into newborn screening programs"
(15). However, the need to monitor MS/MS screening programs on a collaborative basis, with periodic reappraisal of goals
and achievements, is now recognized, and different groups are beginning to work together
to better assess concerns, solutions, and outcomes of MS/MS testing.
The overall consensus of the workshop participants is that the public should
receive accurate information regarding expanded and comprehensive newborn screening
and the evolving knowledge regarding its strengths and weaknesses. State programs
that are ensuring universal opportunity, quality control, tracking, and follow-up should
continue without interruption. Additional disorders other than metabolic disorders
detected by MS/MS are included in comprehensive screening (e.g., congenital adrenal
hyperplasia, cystic fibrosis, sickle cell disease, and biotinidase deficiency). Expansion of
screening should include the more common disorders that are available in certain programs
Work Group Proposals
As a result of information shared at the 2000 workshop and efforts of the work
group, the following overall proposals are offered:
State and territorial health agencies should
consider using MS/MS and other expanded newborn screening technologies
and actively participate in future workshops because these agencies are the
direct sources of funding and regulations for prevention efforts. As research
and reported data grow regarding MS/MS for newborn screening, public
health agencies will want to access this technology either directly or through
regional agreements. Staying current with technological developments will be critical
for policymakers and program managers.
anticipate difficulties in implementing MS/MS testing because the
technology requires new, expanded, and expensive resources. Those resources include
investments in equipment;
expanded information technology support for interpretation,
reporting, tracking, and outcome evaluation;
staff training in clinical and laboratory aspects of detectable disorders;
access to reference laboratories for confirmation of diagnosis; and
assurance of access to adequately skilled clinicians for treatment
involve health-care practitioners, laboratory directors, birthing hospitals,
parents, lay advocacy groups, and third-party payers in a collaborative effort to plan
and define the state and territorial programs and obtain the legal authority
and funding necessary for implementation.
consider contracting with other state laboratories, private laboratories,
or academic medical centers for laboratory services that are too expensive for
local program resources. Alternatively, information could be provided to
health-care practitioners, hospitals, and parents explaining the options for
supplemental testing services, and state/territorial program staff could facilitate access to
these services. States that contract for laboratory or other services should retain
the functions of quality assurance and monitoring (e.g., speed and accuracy of
testing and reporting, tracking, and ensuring quality clinical follow-up).
Federal health agencies should
provide leadership and support to assist states and territories in
implementing MS/MS technology. An effective model for federal involvement in
newborn screening is the assistance that was provided to states and territories
when implementing sickle cell disease testing. A 1986 National Institutes of
Health consensus development conference (39) recommended sickle cell
disease screening for newborns. Congress then appropriated $8 million for the
Special Projects of Regional and National Significance program administered by
HRSA. Using that funding, HRSA awarded grants to enhance the
screening infrastructure. States then reviewed the scientific information,
appropriated funding, and added sickle cell disease to their newborn screening programs.
CDC, in cooperation with HRSA, developed a quality-assurance program and
funded studies for effectiveness evaluation of sickle cell screening methods
support a national MS/MS screening work group with three subcommittees,
a laboratory methods group to address standards, quality assurance,
and methodologic improvements;
a clinical group to define the detectable disorders, available interventions,
and requirements for metabolic diagnosis and management; and
an epidemiologic group to design and implement an evaluation effort
that would include collecting data regarding effectiveness of different
screening policies, disorder prevalence and trends, long-term outcomes, and
cost-effectiveness and cost-benefit.
Representatives from the American Academy of Pediatrics, American College
of Medical Genetics, American Public Health Association, Association of Public
Health Laboratories, Association of State and Territorial Health Officers, National
Newborn Screening and Genetics Resource Center, CDC, and HRSA should
actively participate in workshop, work group, and subcommittee activities.
sponsor long-term epidemiologic studies of MS/MS screening to document
the natural history of metabolic diseases, establish data collection protocols,
and evaluate MS/MS technology's impact.
provide resources to support state and local staff training in MS/MS
analytic techniques and efforts to provide analytical standards and proficiency testing.
provide fiscal and technical support for long-term follow-up, large-scale
data sharing, or development of laboratory quality-assurance programs to
prevent MS/MS applications of mixed quality and effectiveness resulting
from independent and isolated actions.
The work group members acknowledge the contributions of the following persons
for their ideas and discussion that guided the content and preparation of this report: Shu
H. Chaing, Ph.D., Department of Health and Human Services, Raleigh, North Carolina; Jannine
D. Cody, Ph.D., Genetics Alliance, University of Texas Health Science Center at San Antonio,
San Antonio, Texas; Arthur F. Hagar, Ph.D., Louisiana Department of Health and Hospitals,
New Orleans, Louisiana; Kristine K. Hanson, M.S., University of Wisconsin, Madison,
Thomas M. Hickey, Ph.D., South Carolina Department of Health and Environmental
Control, Columbia, South Carolina; Michael E. Jackson, Ph.D., PerkinElmer Life Sciences
(Wallac), Norton, Ohio; David C. Jinks, Ph.D., Minnesota Department of Health Laboratory,
Minneapolis, Minnesota; Celia I. Kaye, M.D., Ph.D., Department of Pediatrics, University of Texas
Health Science Center at San Antonio, Texas; Timothy H. Lim, Ph.D., CDC, Atlanta, Georgia; David
S. Millington, Ph.D., Duke University Medical Center, Research Triangle Park, North
Carolina; Michael R. Morris, Ph.D., Micromass UK, Ltd., Manchester, United Kingdom; Patricia K.
Mullaley, National Coalition for PKU and Allied Disorders, Mansfield, Massachusetts; Edwin W.
Naylor, Ph.D., Neo Gen Screening, Inc., Bridgeville, Pennsylvania; Joerg N. Pirl, Ph.D., Illinois
Department of Public Health, Chicago, Illinois; Rodney J. Pollitt, Ph.D., Children's Hospital,
Sheffield, United Kingdom; William J. Rhead, M.D., Ph.D., Medical College of Wisconsin, Elm
Grove, Wisconsin; Charles R. Roe, M.D., Baylor University Medical Center, Dallas, Texas; John
E. Sherwin, Ph.D., California Department of Health Services, Berkeley, California; Donald
L. Simmons, Ph.D., University of Iowa Hygienic Laboratory, Des Moines, Iowa;
Lawrence Sweetman, Ph.D., Baylor University Medical Center, Dallas, Texas; Sophia S. Wang, Ph.D.,
CDC, Atlanta, Georgia; Bridget Wilcken, M.B., Ch.B., New Children's Hospital, Parramatta,
Australia; and Thomas H. Zytkovicz, Ph.D., New England Newborn Screening Program, Jamaica
We also thank Sue Triesch and Donna C. Williams, National Newborn Screening and
Genetics Resource Center, Austin, Texas, for their administrative services; Carol J. Bell for her
spirited dialogue and assistance; and Wanda E. Whitfield for her graphics support.
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Newborn Screening and Genetics Resource Center for organizing, hosting, and supporting
the 2000 workshop along with their partners, the Association of Public Health Laboratories,
Great Lakes Regional Genetics Group, Institute for Metabolic Diseases, Micromass UK, Ltd.,
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* Newborn is defined as an infant aged
** The proposals in this report are based on conclusions derived by participants in the
plenary and work group sessions held during the workshop.
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