By Harvey L. Levy, M.D.
The content in this publication was current at the time it was published, but it is not being updated. The publication is provided for historical purposes only.
The foundation of newborn screening for phenylketonuria (PKU) was laid by Fölling, who identified PKU as a biochemical cause of mental retardation (Fölling, 1934), and 20 years later by Bickel, who developed a phenylalanine (Phe)-restricted diet that could prevent the mental retardation (Bickel, Gerrard, Hickmans, 1954). Less than 10 years after the diet became known, Guthrie developed a bacterial assay for Phe so that newborns with PKU could be identified for optimal dietary benefit (Guthrie, Susi, 1963). A collaboration between Guthrie and MacCready, a physician who directed the diagnostic laboratories of the Massachusetts Department of Public Health, resulted in the application of Guthrie’s test to spots of blood routinely collected from the heel of newborn infants at nursery discharge and dried on filter paper. Screening in Massachusetts was quickly successful, producing the unexpectedly large number of 9 cases of PKU among the first 53,000 infants tested (MacCready, 1963). Very soon, States passed laws mandating screening for PKU, and today all infants in the United States are being screened (Levy, 1973).
State laws requiring screening for PKU had the unfortunate consequence of leading to a separate screening program in each State rather than to a national program or regional programs (Levy, Albers, 2000). Currently, there are only two truly regional programs: (1) the Northwest Regional Program in which the Oregon Public Health Laboratory also screens specimens from Nevada, Alaska, Wyoming, and Montana, and (2) the New England Regional Program in which the Massachusetts Public Health Laboratory also screens specimens from Maine, Vermont, New Hampshire, and Rhode Island. Smaller pockets of "regionalization" also exist (e.g., Mississippi specimens are screened in Tennessee). For the most part, however, each State independently determines screening policies and conducts screening, leading to inefficiencies and substantial variations in quality (Levy, Albers, 2000).
The screening test is usually performed in a State laboratory within a health department. There are two notable exceptions. In California, the screening test is performed in eight medical center laboratories under contract to the State, and Pennsylvania contracts its screening to Neo Gen Screening, Inc., of Pittsburgh, a large private newborn screening laboratory (Chace, Naylor, 1999).
Guthrie’s bacterial assay remains the most frequently used PKU screening test in the United States (Dougherty, Levy, 1999). This assay is semiquantitative, interpreted by visually comparing the diameter of the bacterial growth zone around a disk from the newborn’s blood specimen with the diameters of growth zones around standards that contain known amounts of Phe (Kim, Levy, 1998). Thus, an infant’s specimen surrounded by bacterial growth zone appearing to have the same diameter as the one around a standard disk containing 4 mg/dL of Phe is assigned the Phe value of 4 mg/dL. A specimen surrounded by a growth zone with slightly smaller or slightly larger diameter is said to have a Phe level of 2 to 4 mg/dL or 4 to 6 mg/dL, respectively. Aside from its lack of quantification, the bacterial assay can only be manually performed. These limitations led some programs to use a quantitative fluorometric assay, which provides a precise value for Phe (e.g., 3.8 mg/dL) and which can be automated (McCaman, Robins, 1962; Hill, Summer, Pender, et al., 1965). In California, this assay has been combined with a fluorometric assay for tyrosine in a two-channel approach that increases its specificity for PKU by providing a Phe-to-tyrosine ratio as well as a Phe level. Tandem mass spectrometry (MS/MS), a single quantitative and automated assay that covers more than 20 disorders, includes very specific and sensitive coverage for PKU (Chace, Millington, Terada, et al., 1993; Chace, Sherwin, Hillman, et al., 1998). Currently, several programs use a fluorometric assay instead of the bacterial assay, and two large programs (Neo Gen Screening, Inc., and New England Newborn Screening Program) use MS/MS.
The effectiveness of screening for PKU incorporates sensitivity and specificity of the method as well as quality of the laboratory performance. In all of these areas, PKU screening has been quite effective. The vast majority of infants with PKU born in the United States have been detected by screening, confirmed, and treated with diet. Thus, although the fluorometric and MS/MS methods are somewhat more sensitive than bacterial assay, this does not appear to be a factor in the relative reliabilities of the methods (Bell, 2000). However, MS/MS displays an amino acid profile or pattern rather than only a Phe level, thus providing greater specificity (fewer false positive results) than either of the other two methods.
Despite a good record on effectiveness, there is much room for improvement. Lack of effectiveness, as determined by missed cases, has largely been the result of poor laboratory or program performance. The reasons have included failure to obtain the blood specimen or collecting an inadequate specimen, errors in the laboratory (e.g., misreading the bacterial assay), reporting abnormal results as normal, and failure to follow up an abnormal result (Holtzman, Slazyk, Cordero, et al., 1986). These missed cases have often occurred in relatively small newborn screening laboratories. This could be remedied by changing the structure of newborn screening from the present largely state-by-state approach to regionalization throughout the United States.
Stored Specimens and Ethical Concerns
Most (75 percent) of the screening programs store the newborn dried blood specimens, the length of time ranging from several months to as long as 25 years (McEwen, Reilly, 1994). These stored specimens have been very valuable in assessing the quality of newborn screening performance by allowing for retesting when a child not identified by screening is later found to have PKU. In virtually all of these instances, the retested result has been a marked increase in Phe rather than the normal result originally reported, demonstrating laboratory error (Levy, Albers, 2000). Stored specimens have also been used for metabolic research and to assess new technologies (Levy, Albers, 2000).
Until recently, storage of the newborn specimens generated little concern. This has now dramatically changed because of their potential use for DNA testing. Since they represent entire populations of infants, these specimens have been called "DNA banks" (McEwen, Reilly, 1994). The concerns include use of the specimens without consent to identify children with untreatable diseases, particularly those that might not be clinically expressed until many years later. Research in which the identity of the infant is removed from the stored specimen (anonymous studies) has generated much less controversy. The current issue of stored specimens, therefore, centers on control of their use and whether they should be used with retention of identifiers.
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