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The Guthrie test, also known as the Guthrie bacterial inhibition assay, is a medical test performed on newborn infants to detect phenylketonuria, an inborn error of amino acid metabolism. The test has been widely used throughout North America and Europe as one of the core newborn screening tests since the late 1960s. In recent years it is gradually being replaced in many areas by newer techniques such as tandem mass spectrometry that can detect a wider variety of congenital diseases. The Guthrie test is named after Robert Guthrie, an American bacteriologist and physician, who devised it in 1962.
Theory and methodEdit
The Guthrie test is a semiquantitative assay designed to detect elevated blood levels of the amino acid phenylalanine, using the ability of phenylalanine to facilitate bacterial growth in a culture medium with an inhibitor.
A drop of blood is usually obtained by pricking the heel of a newborn infant in a hospital nursery on the sixth or seventh day of life (end of the first week). The blood is collected on a piece of filter paper and sent to a central laboratory. A small disk of the filter paper is punched out and placed on an agar gel plate containing Bacillus subtilis and B-2-thienylalanine. Each gel holds 60-80 disks. The agar gel is able to support bacterial growth but the B-2-thienylalanine inhibits bacterial growth. However, in the presence of extra phenylalanine leached from the impregnated filter paper disk, the inhibition is overcome and the bacteria grow. Within a day the bacterial growth surrounding the paper disk is visible to the eye. The amount of growth, measured as the diameter of the colony, is roughly proportional to the amount of phenylalanine in the serum. The result is read by comparing the diameter of each sample disk's colony to the colonies of a series of reference disks with standard phenylalanine content included on each large plate.
Interpretation of resultsEdit
The Guthrie assay is sensitive enough to detect serum phenylalanine levels of 180-240 μmol/L (3-4 mg/dL). In healthy normal people, phenylalanine levels are usually under 120 μmol/L.
When an elevated level of phenylalanine is detected, the laboratory notifies the infant's practitioner, who explains the result to the family and arranges testing to determine the cause of the high phenylalanine levels.
There are a number of causes of hyperphenylalaninemia, and less than 10% of the positive results obtained by the screening program are confirmed as due to phenylketonuria (PKU). There are other metabolic diseases that can produce hyperphenylalaninemia, but false positive results can also occur due to unexplained mild, transient elevations, prematurity, parenteral nutrition, or contamination of the filter paper specimen. False negative results can be produced by antibiotics in the blood sample. Misleadingly low results can also occur if a sample is taken too soon after birth, since phenylalanine levels rise steadily with age and protein feeding.
The sample for guthrie test in the UK is analyzed for the following conditions:
- Phenylketonuria all UK areas.
- Congenital hypothyroidism all UK areas.
- Cystic Fibrosis most UK areas.
- MCAD Deficiency fewer UK areas.
- Sickle Cell Disease fewer UK areas.
The sample for the Guthrie test in New Zealand is analyzed for the following seven conditions:
- Maple Syrup Urine Disease (MSUD)
- Biotinidase deficiency
- Congenital Adrenal Hyperplasia
- Cystic Fibrosis
- Congenital Hypothyroidism
Phenylketonuria (PKU) was one of the earliest recognized inborn errors of metabolism. In populations of European ancestry, it affects about 1 in 13,000 infants. In 1934, Følling discovered that restricting phenylalanine could improve the neurologic function of children with PKU, and within a few years it was established that a diet low in protein from infancy could prevent the mental retardation. Unfortunately, PKU was often not detected until significant brain damage had occurred. The ferric chloride test was unreliable in newborns and even by the 1950s there was no reliable, practical method for mass screening and detection of this disease before harm had occurred.
Robert Guthrie (1916-1995), a bacteriologist and physician at the Women and Children's Hospital of Buffalo, New York, reported in 1963 the initial version of this assay. Within a decade, newborn screening programs using this test were established in most of the United States and many other countries.
- Guthrie R, Susi A (September 1963). A simple Phenylalanine method for detecting Phenylketonuria in large populations of newborn infants. Pediatrics 32: 338–43.