Dr. Robert Guthrie introduced the first newborn screening test in the United States for phenylketonuria (PKU), a progressive and fatal disease of young children, in the early 1960s. While the use of the Guthrie card to collect the heel stick blood for newborn testing is well known to laboratorians and clinicians today, the story of Dr. Guthrie’s implementation of the first newborn screening for PKU may not be.
Dr. Guthrie was born in Marionville, MO, in 1916. He grew up in Minnesota, where he earned his medical degree in 1942 and his PhD in bacteriology in 1946 at the University of Minnesota. For the first 12 years of Dr. Guthrie’s career, he was a principal cancer scientist at the Roswell Park Cancer Institute in Buffalo, NY. During this time, Dr. Guthrie’s second son John was born mentally handicapped (1947) and his 15-month-old niece was diagnosed with PKU (1958). By Dr. Guthrie’s own account, it was the birth of his second son John that motivated him to pursue research aimed at preventing mental retardation and developmental disabilities.1,2 It was the birth of his niece that brought to his attention the treatable causes of mental retardation.
PKU is an autosomal recessive genetic disorder characterized by mental retardation due to a deficiency in phenylalanine hydroxylase, an enzyme necessary to metabolize the essential amino acid phenylalanine to tyrosine (Figure 1). Without phenylalanine hydroxylase, phenylalanine is converted to neurotoxic phenylpyruvic acid. In PKU patients, phenylpyruvic acid accumulates and becomes toxic to the brain, leading to developmental defects and mental retardation. While PKU was discovered in 1935 by Asbjorn Fölling, its diagnosis through the early 1960s was performed by detecting phenylpyruvic acid in urine using a reaction with ferric chloride. While this test was accurate, it was not sensitive enough to detect phenylpyruvic acid until irreversible brain damage had occurred. This was particularly unfortunate because by the 1950s it was known that treatment of PKU with a low-phenylalanine diet could avoid much of the associated neurotoxicity.
By 1957, Dr. Guthrie had become very active in the local Buffalo Chapter of the New York State Association for Retarded Children. As vice president, Dr. Guthrie invited Dr. Robert Warner, director of the newly established Children’s Rehabilitation Center at Buffalo Children’s Hospital, to speak at the organization’s monthly meeting. Dr. Warner’s work on treating children with developmental disorders caught Dr. Guthrie’s attention.2 In subsequent meetings with Dr. Warner, Dr. Guthrie discussed his interest in preventing mental retardation due to inborn errors of metabolism. While still working at the Roswell Park Cancer Institute, Dr. Guthrie was introduced to the need for a more simplified, rapid, and accurate measure of phenylalanine levels in the blood of PKU patients by Dr. Warner.2 Both Dr. Warner and Dr. Mitchell Rubin, chief of pediatrics at the children’s hospital, later persuaded Dr. Guthrie to transfer from the cancer institute to the Buffalo Children’s Hospital in 1958. At the suggestion of Dr. Rubin, Dr. Guthrie set out to develop a new method to monitor phenylalanine. By modifying the bacterial tests he had used in his cancer research, used to detect specific circulating substances in cancer patients, he developed a technique to monitor PKU levels using the same principles of competitive inhibition.2
Metabolic basis of phenylketonuria (PKU). The underlying defect in PKU is a deficiency in the enzyme that converts phenylalanine to tyrosine (phenylalanine hydroxylase), as indicated by the red X. The resulting phenylpyruvic acid builds up over time and becomes toxic to brain development and leads to mental retardation.
The method to detect increased levels of phenylalanine using bacterial inhibition was simple: A standard culture of Bacillus subtilis was incubated on agar in the presence of an antagonist of phenylalanine (B-2-thienylalanine), which prevents the bacteria from growing.3 When blood soaked filter paper discs (cut from the dried blood spots) are placed on the agar, the presence of phenylalanine overcomes the growth inhibition due to the lack of phenylalanine, allowing the determination of excess phenylalanine to be made based on the amount of bacterial growth.3 While this test was developed to monitor the blood phenylalanine in treated children on a low phenylalanine diet, it soon became apparent that it could be utilized as a screening mechanism to prevent the developmental delay and retardation in PKU patients. The original test utilized filter paper soaked with serum; however, it was apparent this would be a difficult specimen to obtain for routine newborn screening because it needed to be processed to get serum. Dr. Guthrie then realized that whole blood collection from an infant heel stick collected with filter paper might work as well. And it did.
A trial PKU screening using this methodology was first arranged in 1960 to test children in the Newark State School for the mentally retarded.4 Filter-paper specimens of blood (Guthrie cards) were collected from the residents and tested with the bacterial inhibition assay of Dr. Guthrie’s design for phenylalanine. The test successfully detected all of the previously confirmed PKU cases as well as four previously undiagnosed by urine screening.4 In 1961, Dr. Guthrie and his laboratory began receiving filter paper specimens of blood from newborn infants in two Jamestown hospitals. With the demand evident, Dr. Guthrie received funding from the United States Children’s Bureau to try screening on a national basis for PKU in newborns.4 This gave Dr. Guthrie the resources to establish a factory within a small house next to the Buffalo Children’s Hospital to prepare material for the PKU screening test. Within two years, 400,000 infants had been tested from 29 states, resulting in no less than 39 positive cases of PKU.2,5,6 Newborn screening for PKU identified an incidence of almost one in 10,000 births in the United States.2 Importantly, no cases were missed by this new method of screening, allowing for definitive therapy to be given to all identified patients before neurotoxicity set in.2
Impressed by Dr. Guthrie’s successes, and driven by Dr. Guthrie’s own determination, the National Association for Retarded Children lobbied for a legally mandated PKU screening throughout the mid 1960s. In 1967, 37 states passed laws mandating PKU screening in all newborns; today, all 50 states have such laws. The greatest success of the development of PKU newborn screening is that it precipitated people to ask whether other diseases may be prevented through similar and parallel early detection methods. It soon became clear that they could, and by the late 1960s, newborn screening for genetic diseases became a part of infant health care in the United States. Most newborn screening programs in the United States now use tandem mass spectrometry (MS/MS) as the principal tool to analyze blood spots. Before tandem mass spectrometry, separate assays (eg, bacterial inhibition assays) were needed for each possible metabolic disorder, in addition to tests for endocrine and hemoglobin disorders, to name a few. The menu of diseases identified by newborn screening continues to expand today, with cystic fibrosis most recently added to the diseases screened for by most states.
Dr. Robert Guthrie died on June 23, 1995, at the age of 78. He was married to his wife for 53 years, with whom he had six children.7 Expanded newborn screening programs based on Dr. Guthrie’s blood collection system for early detection of treatable diseases continues today as the stepping stone for improved children’s health in the United States and the world.
Suggested Reading: Robert Guthrie. The PKU Story. Crusade Against Mental Retardation by Jean Holt Koch. Hope Publishing House, Pasadena, CA.
. The inhibition assay: Its use in screening urinary specimens for metabolic differences associated with mental retardation. Proceedings of the London Conference on The Scientific Study of Mental Deficiency, 1960. ed. RichardsBW, May & Baker Ltd (Dagenham) 1962;2:672.