Potential and pitfalls of NBS, and the reference lab's role.(newborn screening)(Cover story)

CONTINUING EDUCATION

To earn CEUs, see test on page 22.

LEARNING OBJECTIVES

Upon completion of this article, the reader will be able to:

1. To become aware of the morbid condition requirements for newborn screening (NBS).

2. To become familiar with the different methodologies used to screen for genetic disorders, particularly tandem mass spectrometry.

3. To know the limitations of NBS testing.

4. To learn what factors can cause false-positive alerts for amino acids on NBS.

5. To become aware of the commonly measured acylcarnitines and their associated disorders.

6. To be aware of the pitfalls in the interpretation of acylcarnitine profiles by NBS labs when evaluating fatty-acid metabolism disorders.

7. To learn that interlaboratory comparison of results for acylcarnitine analysis is severely limited.

8. To learn what needs to be done to optimize cutoff levels for various analytes for identifying patients that warrant further investigation.

9. To gain an understanding of the short and long term cost involved in false-positive NBS results.

**********

Among the most notable advancements in public health in the 21 st century has been the expansion of newborn screening (NBS) for the early detection and treatment of heritable disorders. Breakthroughs in technology, particularly the use of tandem mass spectrometry (MS/MS), have allowed for the development of fast and inexpensive protocols to quantify amino, organic, and fatty-acid metabolites as markers for inborn errors of metabolism on a scale amenable to population screening. Individually, most of these disorders are quite rare, but taken together, they represent a significant proportion of the identifiable genetic conditions with a strong potential for mental retardation, morbidity, and mortality.

[ILLUSTRATION OMITTED]

There is no existing federal mandate regarding state obligations for newborn screening. An up-to-date summary of the current policy for individual states, including diseases screened for and financing strategies, can be found at http://genes-r-us.uthscsa.edu.

A formal set of recommendations to Congress for a universal core and supplemental panel of disorders to be screened for by all states was made by the Health Resources and Services Administration and the NBS Committee of the American College of Medical Genetics in 2005, though compliance is currently voluntary. In Ohio, an effective screening strategy has been implemented for all but one of the targeted conditions.

To be suitable for newborn screening, a disorder should meet the minimal criteria as outlined in Table 1. Paramount in this discussion is the understanding that NBS is not meant to be diagnostic in itself. For some disorders, repeating the NBS test following an "alert" is inappropriate and potentially misleading, and urgent consultation with the state or regional metabolic-disease consultant is strongly recommended to determine the most efficient approach to evaluate a suspected disorder. It is the role of the reference laboratory to provide confirmatory testing or recommendations for more specialized testing procedures. A detailed understanding of the potential and the pitfalls of follow-up testing is critical to the rapid resolution of NBS "alerts."

MS/MS usefully innovative

NBS laboratories utilize a variety of technologies to screen for genetics disorders, though all rely on the filter-paper blood spot as the biological source for analytic testing. High-performance liquid chromatography (HPLC) and isoelectric focusing are the principal methodologies for identifying sickle-cell disease and other hemoglobinopathies. Direct analysis of enzyme activity is used in testing for galactosemia (galactose-1-phosphate uridyltransferase deficiency) and biotinidase deficiency. Immunofluorometric techniques are commonly employed in screening for congenital hypothyroidism (TSH) and 21-hydroxylase deficiency (17-hydroxyprogesterone), as well as cystic fibrosis (immunoreactive trypsinogen, or IRT). Targeted DNA-mutation testing has been proven useful as an adjunct to IRT in identifying newborns most at risk for cystic fibrosis. The commercial use of tandem mass spectrometry, however, has been the single most useful innovation introduced for NBS in the last 10 years, largely as a consequence of its ability to simultaneously assay a large number of critical analytes as markers for rare metabolic disorders in an extremely rapid, reproducible, and inexpensive fashion.

[ILLUSTRATION OMITTED]

The overall process is a highly specific way for detecting specific substances in complex mixtures. It can be coupled to an HPLC system, which acts as an autosampler and can provide the ability to separate compounds with the same nominal masses using a column. The combined system is called LC-MS/MS. Column-separation techniques involve significantly more processing time per sample, and are of limited clinical utility in an NBS laboratory that may be analyzing 500 or more samples per day. To save time and expense--and to be practical for large-scale screening--NBS blood-spot-sample derivatized eluents are injected directly into the tandem mass spectrometer.

In the analysis, the metabolites are typically extracted from the dried blood sample on the filter-paper disc or from 3.1 [micro]l of serum or plasma, chemically derivatized, and subjected to analysis by electrospray tandem mass spectrometry. The function of a tandem mass spectrometer is to:

1. produce ions from the compounds in the sample under analysis;

2. select PRECURSOR (PARENT) ions according to their mass in the first analyzer of the instrument;

3. fragment the mass-selected PRECURSOR (PARENT) ions by colliding them with argon gas in the collision cell to give PRODUCT (DAUGHTER) ions--a process called COLLISION-INDUCED DISSOCIATION (CID); and

4. analyze the PRODUCT (DAUGHTER) ions according to their mass in the second analyzer of the tandem instrument.

MS/MS' limited effectiveness in certain areas

In the NBS setting, MS/MS is employed to screen for disorders of amino, organic, and fatty-acid metabolism, as well as several urea-cycle abnormalities. It is equally important to emphasize what it cannot do. MS/MS has proven to be ineffective in reliably identifying deficiencies in the urea-cycle enzymes ornithine transcarbamylase (OTC) and carbamyl phosphate synthetase (CPSI) through the quantitation of citrulline, though is considerably more useful when detecting elevations in this analyte in newborns with a deficiency in argininosuccinic acid synthetase (ASAS) or argininosuccinic acid lyase (ASAL). Glycine has not been shown to be so consistently elevated at 24 hours of age as to make this useful in screening for nonketotic hyperglycinemia, or NKH. Preliminary clinical research studies suggest that quantitation of tyrosine in the first two days of life is extremely unreliable in ascertaining infants with tyrosinemia type 1 (fumarylacetoacetase deficiency). It is incumbent upon physicians to know the limitations of NBS testing, and not to blindly assume that a normal comprehensive profile effectively excludes an inborn error of metabolism.

Amino acids quantitated at 24 hours of age for NBS using MS/MS typically include citrulline, arginine, leucine/isoleucine, valine, methionine, tyrosine, and phenylalanine. Cutoffs to establish "alert" notifications vary between laboratories, and are commonly set between three and six standard deviations (SDs) above the newborn population mean. Since tandem mass spectrometers separate and quantify compounds of interest based on their molecular weight (or more precisely, their mass:charge ratio), amino acids with identical weights cannot be separated using MS/MS alone. Leucine, isoleucine, alloisoleucine, and hydroxyproline all have molecular weights of about 131 and are quantitated together for this reason. Therefore, follow-up testing by the reference lab requires the use of ion-exchange column chromatography rather than MS/MS when maple-syrup urine disease is suspected. Further, an amino-acid…