June 2011: Volume 37, Number 6
The Tandem Mass Spectrometry Revolution
By Suzanne T. Kotkin-Jaszi, DrPH and John E. Sherwin, PhD
Newborn screening, the process of testing newborn babies for treatable genetic, metabolic, endocrinologic, and hematologic diseases, is required by state laws and performed on newborns as part of state public health programs. Today, all 50 states offer newborn screening services and virtually all (>99%) of the more than 4 million babies born in the U.S. each year are screened for conditions such as phenylketonuria (PKU), congenital hypothyroidism, galactosemia, and hemoglobinpathies. These programs have expanded rapidly in the past decade due to advances in science, consumer advocacy, and financial incentives such as federal support of sickle cell testing. Most states manage their own newborn screening programs, although some run programs via regional partnerships with others, while the District of Columbia and Pennsylvania contract for testing services from private laboratories.
Tandem mass spectrometry (MS/MS) has been instrumental to increasing the number of conditions checked by newborn screening programs. In 1995, a few laboratories began using a MS/MS method developed by Millington and Chace and adapted by Naylor for newborn screening (1–3). Based on the success of these programs, the Health Resources and Services Administration (HRSA) issued a report in 2005 from the American College of Medical Genetics (ACMG) supporting the use of MS/MS in newborn screening programs as a means to expand screening capabilities and include more conditions (4). Since then, state programs have sped up their adoption of MS/MS, and today, all states use MS/MS to screen for at least 31 conditions, and as many as 50 depending on the state.
This article will describe how labs employ MS/MS in newborn screening, why clinical laboratorians should be knowledgeable about newborn screening programs, and look ahead to emerging issues in the field.
Overview of Inborn Errors of Metabolism
When a baby is born with an amino acid metabolism disorder, it lacks or produces insufficient levels of specific enzymes needed to convert fat to energy or to breakdown amino acids. In the latter instance, such inborn errors of metabolism (IEM) cause amino acids to accumulate, and, if undetected and untreated, to become toxic. For example, infants with a specific DNA mutation lack the enzyme phenylalanine hydroxylase and cannot metabolize the essential amino acid, phenylalanine. MS/MS effectively screens for this metabolic disease, known as PKU, and many other IEMs, including amino acid, organic acid, and fatty acid oxidation disorders.
Newborn screening for metabolic disorders began in 1961, when Robert Guthrie developed a simple, cost-effective screen-ing test for PKU. States rapidly adopted the test, and by the late 1960s, the practice of screening for PKU had spread to almost all 50 states and some developing countries. The number of IEMs included in newborn screening gradually expanded over the years as researchers developed new tests, including ones for primary congenital hypothyroidism, sickle cell disease, amino acidopathies, and galactosemia.
Inborn Errors of Metabolism: Incidence
The birth of a baby with an IEM is not a rare event. In the U.S., the overall incidence of IEMs is estimated to be 1 in 4,000 live births (5). The incidence and frequency of individual diseases varies, however, depending on the racial and ethnic composition of the population and the extent to which screening programs are available to help clinicians make a differential diagnosis.
The authors of a draft ACMG report estimate the incidence of PKU alone in the U. S. to be 1 in 12,000 live births, with the combined incidence for all amino acidopathies as high as 1 in 6,000 (6). Not included in these estimates are other IEMs, such as organic acid disorders, some urea cycle disorders, and congenital lactic acidemias that may require amino acid analysis for diagnosis and monitoring of treatment. Table 1 provides the incidence rates for several IEMs reported in a study conducted in British Columbia, Canada (7).
Incidence of Inborn Errors of Metabolism
|Type of Inborn Error
|Disease involving amino acid disorders, organic acid, primary lactic acidosis, galactosemia, or a urea cycle disease
||24 per 100,000 births
||1 in 4,200|
|Lysosomal storage disease
||8 per 100,000 births
||1 in 12,500|
||Approximately 3 to 4 per 100,000 births
||1 in 30,000|
|Respiratory chain-based mitochondrial disease
||Approximately 3 per 100,000 births
||1 in 33,000|
|Glycogen storage disease
||2.3 per 100,000 births
||1 in 43,000|
|This data is based on a study in British Columbia, Canada that estimated the overall incidence rate for inborn errors of metabolism at 70 per 100,000 live births or 1 in 1,400 births. The authors also suggested that it is difficult to generate accurate incidence and prevalence data because of under-diagnosis and the fact that some patients will not show signs and symptoms of disease until after the time period of the survey (7).|
The Analytical Procedure
In simple terms, mass spectrometers are analytical instruments that separate and quantify molecular ions based on their mass/charge (m/z) ratios. As the name implies, MS/MS instruments usually consist of two mass spectrometers separated by a reaction chamber or collision cell. A complete description of how these instruments work is beyond the scope of this discussion, but suffice it to say they detect and quantitate compounds in body fluids and are therefore ideal for newborn screening.
Soon after birth, hospital staff obtains capillary blood from newborns by pricking their heels and collecting a drop of blood on a special filter paper known as a Guthrie card. Initially, laboratories using MS/MS for newborn screening eluted infants’ samples from the blood spots using methanol followed by derivatization with butanolic HCl, evaporation, and then reconstitution with an appropriate organic solvent for introduction into the spectrometer. While many programs still use this technique, an increasing number of laboratories are changing to a direct elution and injection technique, because it both saves sample preparation time and eliminates a caustic chemical, butanolic HCl, from the laboratory.
For the actual analysis, a technician takes a 3-mm punch from the dried blood spot and then extracts it with methanol by agitating for about 15 minutes. The next steps involve adding a solution of internal standards and evaporating the extraction. The technician then butylates the residue and analyzes it using MS/MS. Alternatively, some screening programs skip the butylation step and directly inject the eluate into the instrument. This methodology entails eluting the sample with methanol, centrifuging it, and then evaporating the eluate until it is dry. The technician then reconstitutes the eluate with an injection solvent —typically acetonitrile, formic acid, and water—and places it in sealed microtiter plates for the MS/MS analysis.
A More Complete Picture
The development and dissemination of the MS/MS technology for newborn screening has vastly improved detection of IEMs. Table 2 shows the prevalence of eight metabolic disorders identified by MS/MS-based testing of more than 2 million newborns during a 5-year period. In fact, the data reported in both Tables 1 and 2 would not be available without the widespread use of MS/MS technology by newborn screening programs.
Prevalence of Metabolic Disorders that Can Be Identified by MS/MS
The data are derived from MS/MS testing of 2,206,640 newborns between July 2005–June 2009 in the California Genetic Disease Testing Program (unpublished).
MCADD—medium chain acyl CoA dehydrogenase deficiency
SCADD—short-chain acyl-CoA dehydrogenase deficiency
MMA/PA—propionic scidemia (PA) or methylmalonic acidemia (MMA)
VLCADD—very long chain acyl-CoA dehydrogenase deficiency
3-MCC—3-methylcrotonyl-CoA carboxylase deficiency
IVA—isovaleric academia; MSUD- maple syrup urine disease
Today seven states screen for more than 50 IEMs by MS/MS. Furthermore, last year, all states using this technology screened for no fewer than 30 medical conditions (Figure 1). In sum, MS/MS is a cost-effective technology that has allowed simultaneous high-volume screening of newborns for multiple metabolic disorders, including amino acidopathies, fatty acid oxidation disorders, and organic acid disorders.
Facing the Challenges
While adoption of this technology has been rapid and widespread, many critical analytical, laboratory, and policy issues still need to be resolved. On the analytical and laboratory front, these issues include: development and refinement of analyte-specific cutoff values to minimize false-negatives/false-positives; development and adaptation of guidelines across newborn screening laboratories to ensure comparable result quality; improved management of newborn screening programs to ensure they are collaborating with clinical laboratories on shortening turnaround times; and improved communication between clinical laboratories, screening programs, community-based metabolic specialists, and primary care providers to obtain samples and achieve a definitive diagnosis as rapidly as possible.
Newborn screening laboratories set up and operate MS/MS in different ways; therefore, a critical need for the field is developing a consensus on methodological approaches. Doing so will also improve comparability of results across laboratories. In an effort to move towards this consensus, the Clinical and Laboratory Standards Institute recently published a set of guidelines, Newborn Screening by Tandem Mass Spectrometry; Approved Guidelines (www.clsi.org).
Newborn screening programs also need to better understand the natural history and variable presentations of infants identified as potential positives for IEMs by MS/MS. This challenge underscores the fact that successful newborn screening requires a true team effort, not only to identify infants at risk, but also to ensure that these infants receive an appropriate diagnostic evaluation. Babies found to be truly affected require prompt referrals to appropriate medical specialists for ongoing clinical evaluation and medical treatment.
Perhaps the most critical analytical issue facing laboratories is the need for well-developed cutoff values. Published reviews of large-scale newborn screening programs that use MS/MS suggest that clinical laboratories need to pay more attention to developing appropriate cutoffs for many analytes. False-positive results can create extreme anxiety for parents and often require expenditure of limited resources to collect follow-up specimens and perform repeat tests. One suggestion now being discussed in the field is creating different cutoff values for different subpopulations of newborns.
Although adoption of MS/MS for newborn screening clearly has increased the number of IEMs routinely evaluated, many state governments face critical policy decisions about whether to include certain rare metabolic disorders detectable by MS/MS in their state’s program. The 2005 ACMG/HRSA report recommended that 29 conditions be included in a core newborn screening panel. The authors considered these disorders appropriate for all newborn screening programs because screening tests and efficacious treatments exist for them, as well as a substantial body of knowledge about their natural histories. Of these conditions, MS/MS can identify 23, making it the dominant technology of core newborn screening programs.
Public pressure is mounting on state newborn screening programs to screen for more congenital diseases for a variety of reasons, including advances in science, the desire to decrease morbidity and mortality, and marketing efforts by the private sector and other forces. However, other issues are at play beyond the ability of cutting-edge MS/MS technologies to detect additional IEMs. Expansion of newborn screening programs and MS/MS technologies does not depend solely on developing laboratory expertise to collect the sample, operate the MS/MS, analyze the results, and interpret the data. Another crucial factor is the need to train an entire public health team to make the differential diagnosis, ensure timely follow-up, and deliver appropriate medical treatment.
In 2003, the Secretary of the U.S. Department of Health and Human Services chartered the Secretary’s Advisory Committee on Heritable Disorders in Newborns and Children to make recommendation on the most appropriate application of universal newborn screening tests, technologies, policies, guidelines, and standards for effectively reducing morbidity and mortality in newborns and children having, or at risk for, heritable disorders. This group continues to review the published literature for additional IEMs that should be added to newborn screening programs. Future likely candidates include lysosomal storage disorders and mucopolysaccharidoses.
Another key consideration is the cost of expanding newborn screening programs. Currently, the primary financing source is fees paid by third-party payers. A report from the U.S. Government Accountability Office (GAO) estimated that about two-thirds of the funding came from fees and the reminder from federal block grants, specifically the Maternal and Child Health Block, Medicaid, and other state revenues. Fees cover most of the cost of screening, but not treatment and follow-up (8).
Precise data on cost-effectiveness can not be calculated because data on definitive testing volumes is lacking. Schulze and colleagues estimated the cost per procedure by MS/MS at $7.50 per test. They used an observed frequency of 1 in 4,100 newborns to calculate the cost to detect one affected infant at $30,750 dollars. The authors correctly suggest that this cost will be quickly offset by a reduction in medical and hospital expenses of approximately $30,000–$40,000 per year, or $0.5–1.5 million in lifetime medical costs for a pediatric patient with an IEM who is not diagnosed early and treated (9).
Another analysis conducted by Canadian researchers in 2005 suggested that for an MS/MS screening program to be effective, diseases should be grouped into a single bundle; however, screening for all possible diseases in the bundle would not be cost-effective. These researchers suggested that the incremental cost of screening by life-year gained would be optimized by screening for PKU and nine other diseases (10).
On the other hand, technology advancements may make screening for even more disorders feasible. For such expanded screening to be cost-effective and clinically efficacious, however, other resources will be needed. These include the resources for immediate follow-up, rapid timing of confirmatory testing, and specialized treatment. Furthermore, to reduce the anxiety of families with affected infants, clinical laboratories will need to operate both efficiently and effectively and stay closely linked with the metabolic unit treating the child.
It seems clear that the future will bring additional multiplex technologies, including DNA arrays that will be useful in newborn screening programs. Although interpreting the association between a positive DNA microarray result and an infant’s clinical presentation will be a complex challenge, we expect that this technology holds great promise for detecting IEMs.
- Millington DS, Kodo N, Norwood DL, Roe CR. Tandem mass spectrometry: A new method for acylcarnitine profiling with potential for neonatal screening for inborn errors of metabolism. J Inherit Metab Dis 1990;13:321–324.
- Chace DH, Kalas TA, Naylor EW. Use of tandem mass spectrometry for multianalyte screening of dried blood specimens from newborns. Clin Chem 2003;49:1797–1817.
- Naylor EW, Chace DH. Automated tandem mass spectrometry for mass newborn screening for disorders in fatty acid, organic acid, and amino acid metabolism. J Child Neur 1999;14:Suppl(1):S4–S8.
- Health Resources Services Administration, Maternal Child Health Bureau. Newborn Screening: Toward a Uniform Screening Panel and System. 2005. Available online. Accessed July 30, 2010.
- Wikipedia, Inborn errors of metabolism, Available from: http://en.wikipedia.org/wiki/Inborn_error_of_metabolism. Accessed on August 19, 2010.
- American College of Medical Genetics, Technical standards and guidelines for amino acid analysis: draft for member comment. Available online. Accessed on August 19, 2010.
- Applegarth DA, Toone JR, Lowry RB. Incidence of inborn errors of metabolism in British Columbia, 1969-1996. Ped 2000;(105)e10. Available online. Accessed on August 19, 2010.
- U.S. Government Accountability Office, Newborn screening: Characteristics of state programs, March 2003; GAO-03-449. Available online. Accessed on August 19, 2010.
- Schulze A, Linder M, Kohlmuller D, Olgemoller K, et al. Expanded newborn screening for inborn errors of metabolism by electrospray ionization-tandem mass spectrometry: results, outcome, and implications. Peds 2003;111:1399–1406.
- Cipriano LE, Rupar CA, Zaric GS. The cost effectiveness of expanding newborn screening for up to 21 inherited metabolism disorders using tandem mass spectrometry: Results from a decision-analytic model. Value in Health 2007;10:83–97.
Suzanne T. Kotkin-Jaszi, DrPH, is an associate professor of Public Health at California State University in Fresno.
John E. Sherwin, PhD, recently retired from his position as director of laboratory operations for Perkin Elmer, Inc.
Disclosure: Dr. Sherwin has stocks/bonds with Perkin Elmer, Inc.