Dreyngerous 2006838665 HS221/03

Screening test for phenylketonuria
Phenylketonuria (PKU) is a genetic disorder that is characterized by an inability of the body to utilize the essential amino acid, phenylalanine. PKU is due to a recessively inherited defect in which the body cannot use the amino acid phenylalanine properly. Phenylalanine, which comes from all dietary protein, accumulates in the blood. The practice of newborn screening was originally developed to detect PKU. There are a variety of laboratory tests used in screening for PKU. Elevated levels of phenylalanine are most often detected using a bacterial inhibition (Guthrie) assay, a semiquantitative test. Guthrie test  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.  Method: 1. A drop of blood is usually obtained by pricking the heel of a newborn infant in a hospital nursery on the second or third day of life. 2. The blood is collected on a piece of filter paper and mailed to a central laboratory. 3. 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-2thienylalanine 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. 4. Within a day the bacterial growth surrounding the paper disk is visible to the eye. 5. The amount of growth, measured as the diameter of the colony, is roughly proportional to the amount of phenylalanine in the serum. 6. 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 results 1. The Guthrie assay is sensitive enough to detect serum phenylalanine levels of 180-240 μmol/L (3-4 mg/dL). 2. In healthy normal people, phenylalanine levels are usually under 120 μmol/L. 3. When an elevated level of phenylalanine is detected, the laboratory notifies the infant's physician, who explains the result to the family and arranges testing to determine the cause of the high phenylalanine levels.

4. 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 5. false positive results can also occur due to unexplained mild, transient elevations, prematurity, parenteral nutrition, or contamination of the filter paper specimen. 6. False negative results can be produced by antibiotics in the blood sample. 7. 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. Increasingly, tandem mass spectrometry (MS/MS) is being used for newborn screening because this laboratory testing technology substantially increases the number of metabolic disorders that can be detected from dried blood-spot specimens. Tandem mass spectrometry measures many different molecules in a single test. MS/MS detects abnormal levels of amino acids and acylcarnitines. Abnormal elevations of these amino acids may indicate the newborn is at risk for disorders of amino acid metabolism. Free carnitine is a chemical that acts as a transporter of fatty acids into and out of the mitochondria, and is necessary for normal energy metabolism. Acylcarnitines are fatty acids or organic acids that are bound to carnitine. These acylcarnitines vary in size, based on the number of carbon atoms and the type of chemical bonds within the molecule. Abnormal levels of these acylcarnitines can act as markers for certain organic acid and fatty acid oxidation metabolic disorders. Automated ESI tandem MS newborn screening of amino acids and acylcarnitines extracted from a single punch of a dried-blood spot, with stable-isotope-labeled internal standards and derivatization to butyl esters, can be performed with 2–4 min of instrument time. This single test is capable of detecting 20–40 inherited metabolic disorders, depending on which analytes are measured and how different disorders are defined. Using ionization techniques of fast atom bombardment or liquid secondary ionization with tandem MS, they simultaneously determined a large number of acylcarnitines as an acylcarnitine profile. This allowed newborn screening for numerous inherited fatty acid oxidation and organic acid disorders by a single procedure.