Guthrie test - AbsoluteAstronomy.com

The Guthrie test, also known as the Guthrie bacterial inhibition assay, is a medical

Medicine

Medicine is the science and art of healing. It encompasses a variety of health care practices evolved to maintain and restore health by the prevention and treatment of illness....

test performed on newborn infants to detect phenylketonuria

Phenylketonuria

Phenylketonuria is an autosomal recessive metabolic genetic disorder characterized by a mutation in the gene for the hepatic enzyme phenylalanine hydroxylase , rendering it nonfunctional. This enzyme is necessary to metabolize the amino acid phenylalanine to the amino acid tyrosine...

, an inborn error

Inborn error of metabolism

Inborn errors of metabolism comprise a large class of genetic diseases involving disorders of metabolism. The majority are due to defects of single genes that code for enzymes that facilitate conversion of various substances into others...

of amino acid

Amino acid

Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...

metabolism

Metabolism

Metabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...

. The test has been widely used throughout North America and Europe as one of the core newborn screening

Newborn screening

Newborn screening is the process by which infants are screened shortly after birth for a list of disorders that are treatable, but difficult or impossible to detect clinically. Screening programs are often run by state or national governing bodies with the goal of screening all infants born in the...

tests since the late 1960s. In recent years it is gradually being replaced in many areas by newer techniques such as tandem mass spectrometry

Tandem mass spectrometry

Tandem mass spectrometry, also known as MS/MS or MS2, involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages.-Tandem MS instruments:...

that can detect a wider variety of congenital diseases. The Guthrie test is named after Robert Guthrie

Robert Guthrie

Dr. Robert Guthrie was an American microbiologist, the first to use dried blood spot testing, which he first did in the 1960's...

, an American bacteriologist

Bacteriology

Bacteriology is the study of bacteria. This subdivision of microbiology involves the identification, classification, and characterization of bacterial species...

and physician

Physician

A physician is a health care provider who practices the profession of medicine, which is concerned with promoting, maintaining or restoring human health through the study, diagnosis, and treatment of disease, injury and other physical and mental impairments...

, who devised it in 1962.

Theory and method

The Guthrie test is a semiquantitative assay

Assay

An assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...

designed to detect elevated blood levels of the amino acid phenylalanine

Phenylalanine

Phenylalanine is an α-amino acid with the formula C6H5CH2CHCOOH. This essential amino acid is classified as nonpolar because of the hydrophobic nature of the benzyl side chain. L-Phenylalanine is an electrically neutral amino acid, one of the twenty common amino acids used to biochemically form...

, using the ability of phenylalanine to facilitate bacteria

Bacteria

Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...

l growth in a culture medium

Microbiological culture

A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions. Microbial cultures are used to determine the type of organism, its abundance in the sample being tested,...

with an inhibitor.

A drop of blood is usually obtained by pricking the heel of a newborn infant in a hospital

Hospital

A hospital is a health care institution providing patient treatment by specialized staff and equipment. Hospitals often, but not always, provide for inpatient care or longer-term patient stays....

nursery

Nursery

Nursery can refer to:Childcare* Nursery , a room within the house designed for the care of a young child or children* Nursery school, a daycare facility for preschool-age children...

on the sixth or seventh day of life (end of the first week). The blood is collected on a piece of filter paper

Filter paper

Filter paper is a semi-permeable paper barrier placed perpendicular to a liquid or air flow. It is used to separate fine solids from liquids or air.-Properties:Filter paper comes in various porosities and grades depending on the applications it is meant for...

and sent to a central laboratory. A small disk of the filter paper is punched out and placed on an agar gel

Agar

Agar or agar-agar is a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of agarophyte red algae. Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium...

plate containing Bacillus subtilis

Bacillus subtilis

Bacillus subtilis, known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase-positive bacterium commonly found in soil. A member of the genus Bacillus, B. subtilis is rod-shaped, and has the ability to form a tough, protective endospore, allowing the organism to tolerate...

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 results

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

Type I and type II errors

In statistical test theory the notion of statistical error is an integral part of hypothesis testing. The test requires an unambiguous statement of a null hypothesis, which usually corresponds to a default "state of nature", for example "this person is healthy", "this accused is not guilty" or...

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.

Other tests

The term "Guthrie test" is sometimes used in a broader sense to describe dried blood spot testing

Dried blood spot testing

Dried blood spot testing is a method of screening for HIV infection and other conditions using DNA amplification.-History:The concept that capillary blood, obtained from pricking the heel or finger and blotted onto filter paper, could be used to screen for metabolic diseases in large populations...

, which can be used to test for several other conditions. The filter cards are sometimes called "Guthrie cards".

The sample for guthrie test in the UK is analyzed for the following conditions:

Phenylketonuria
Phenylketonuria
Phenylketonuria is an autosomal recessive metabolic genetic disorder characterized by a mutation in the gene for the hepatic enzyme phenylalanine hydroxylase , rendering it nonfunctional. This enzyme is necessary to metabolize the amino acid phenylalanine to the amino acid tyrosine...

all UK areas.
Congenital hypothyroidism
Congenital hypothyroidism
Congenital hypothyroidism is a condition of thyroid hormone deficiency present at birth. Approximately 1 in 4000 newborn infants has a severe deficiency of thyroid function, while even more have mild or partial degrees. If untreated for several months after birth, severe congenital hypothyroidism...

all UK areas.
Cystic Fibrosis
Cystic fibrosis
Cystic fibrosis is a recessive genetic disease affecting most critically the lungs, and also the pancreas, liver, and intestine...

most UK areas.
MCAD Deficiency
Medium-chain acyl-coenzyme A dehydrogenase deficiency
Medium-chain acyl-coenzyme A dehydrogenase deficiency is a fatty acid oxidation disorder associated with inborn errors of metabolism. It is due to defects in the enzyme complex known as medium-chain acyl dehydrogenase and reduced activity of this complex...

fewer UK areas.
Sickle Cell Disease fewer UK areas.

The sample for the Guthrie test in New Zealand is analyzed for 28 conditions:

Amino acid disorders (14 disorders, including PKU and MSUD)
Fatty acid oxidation disorders (9 disorders)
Galactosaemia
Biotinidase deficiency
Biotinidase deficiency
Biotinidase deficiency is an autosomal recessive metabolic disorder in which biotin is not released from proteins in the diet during digestion or from normal protein turnover in the cell. This situation results in biotin deficiency....
Congenital adrenal hyperplasia
Congenital adrenal hyperplasia
Congenital adrenal hyperplasia refers to any of several autosomal recessive diseases resulting from mutations of genes for enzymes mediating the biochemical steps of production of cortisol from cholesterol by the adrenal glands ....
Cystic fibrosis
Cystic fibrosis
Cystic fibrosis is a recessive genetic disease affecting most critically the lungs, and also the pancreas, liver, and intestine...
Congenital hypothyroidism
Congenital hypothyroidism
Congenital hypothyroidism is a condition of thyroid hormone deficiency present at birth. Approximately 1 in 4000 newborn infants has a severe deficiency of thyroid function, while even more have mild or partial degrees. If untreated for several months after birth, severe congenital hypothyroidism...

History

Phenylketonuria

(PKU) was one of the earliest recognized inborn errors of metabolism

Inborn error 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

Protein

Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

from infancy could prevent the mental retardation

Mental retardation

Mental retardation is a generalized disorder appearing before adulthood, characterized by significantly impaired cognitive functioning and deficits in two or more adaptive behaviors...

. Unfortunately, PKU was often not detected until significant brain damage had occurred. The ferric chloride test

Ferric chloride test

The ferric chloride test is used to determine the presence or absence of phenols in a given sample. Enols give positive results as well. The bromine test is useful to confirm the result, although modern spectroscopic techniques are far superior in determining the identity of the unknown...

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

Robert Guthrie

Dr. Robert Guthrie was an American microbiologist, the first to use dried blood spot testing, which he first did in the 1960's...

(1916-1995), a bacteriologist and physician

Physician

at the Women and Children's Hospital of Buffalo, New York

New York

New York is a state in the Northeastern region of the United States. It is the nation's third most populous state. New York is bordered by New Jersey and Pennsylvania to the south, and by Connecticut, Massachusetts and Vermont to the east...

, 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

United States

The United States of America is a federal constitutional republic comprising fifty states and a federal district...

and many other countries.

Theory and method

Interpretation of results

Other tests

History

See also