Newborn Screening Information for Families:
Laboratory Testing Overview

Lab testingOn this page:
Metabolic Disorders
Endocrine Disorders
Hemoglobinopathies
Galactosemia
Biotinidase Deficiency
Cystic Fibrosis
Severe Combined Immunodeficiency
Questions

 


Metabolic Disorders

Metabolic disorders occur when there is a problem with how our body breaks down food into its simpler components: proteins, fats, and carbohydrates. The metabolic disorders included on the newborn screening panel can be grouped into three categories: amino acid disorders, fatty acid oxidation disorders, and organic acid disorders. Amino acid disorders result when there is too much of an amino acid (building block of protein) present in the body. For example, someone with an amino acid disorder like Phenylketonuria (PKU) has too much of the amino acid, phenylalanine. Individuals with PKU need to be on a special diet to prevent health complications. Fatty acid oxidation disorders result when there is a problem with the way the body metabolizes fat, which leads to an accumulation of a compound called acylcarnitines. Someone with a fatty acid oxidation disorder like Medium-Chain Acyl-CoA Dehydrogenase (MCAD) deficiency is not able to properly break down fat into energy. Individuals with MCAD deficiency need to avoid long periods of time without eating and need to be on a special diet to prevent health problems. Organic acid disorders result when there is a problem with how we break down proteins. For example, someone with an organic acid disorder like Glutaric Acidemia, Type 1 (GA1) cannot properly break down protein from the food they eat in order for them to use it for energy and growth. Individuals with GA1 need to be on a special low-protein diet to prevent health complications.

Tandem mass spectrometry is the test used in newborn screening to determine if an infant is at risk for an amino acid, fatty acid oxidation, or organic acid disorder. Scientists use the term "mass" to describe a molecule's weight. Since every molecule has a unique mass, we are able to easily identify the specific molecule present based on its weight. Newborn screening uses a mass spectrometer to identify, analyze, and measure how much amino acids and acylcarnitines are present in a baby's dried blood spot. For example, if you were to grab a handful of coins, you would be able to sort out the dimes, pennies, nickels, and quarters from each other, and you would also be able to count how many of each you have. This is similar to how a mass spectrometer works.

A Tandem Mass Spectrometer is essentially two mass spectrometers that are connected together by a chamber called a collision cell. The first step in tandem mass spectrometry is placing the sample into the mass spectrometer. The first mass spectrometer identifies and measures each molecule present in the blood. For example, it can sort out which molecules are proteins. Once complete, it then enters the collision cell. The collision cell separates the molecule further into multiple fragments. For example, it breaks down a protein into individual amino acids. In the second mass spectrometer, each fragment (amino acid) can then be identified by its mass and electric charge.

In newborn screening, if too much of an amino acid is present, the infant is at risk for an amino acid disorder. If too much acylcarnitines are present, the infant is at risk for a fatty acid oxidation or organic acid disorder. This test can screen for more than 40 disorders with a single dried blood spot.

For certain disorders detected by tandem mass spectrometry, DNA analysis is performed to confirm the result. This test looks only for specific changes (mutations) commonly identified in individuals with the disorder. The disorder name and the mutations tested for are listed below:

Disorder Mutation(s)
Medium-chain acyl-CoA dehydrogenase
(MCAD) deficiency
985A>G
199T>C
Long-chain 3-hydroxyacyl-CoA
dehydrogenase (LCHAD) deficiency
1528G>C
Glutaric acidemia type I A421V
R402W
Propionic acidemia – Beta E168K
1218del14/ins12
1170insT
Methylmalonic acidemia N219Y
G717V
3-methylcrotonyl-CoA carboxylase
deficiency
518insT
Maple syrup urine disease Y438N
(previously known as Y393N)
Isovaleric academia A282V
 

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Endocrine Disorders

Endocrine disorders occur when there is a problem with the amount of hormones (chemical messengers) in our body. The endocrine disorders identified with newborn screening are Congenital Adrenal Hyperplasia (CAH) and Congenital Hypothyroidism (CH). Time-Resolved Fluoroimmunoassays are used to test for these disorders.

Fluoroimmunoassays use two different antibodies (specialized proteins) that bind to a molecule of interest (for example a hormone) present in the blood. One of the two antibodies used in the test contains a fluorescent label on it. Fluorescent light is released as a result, which can be measured to determine how much of the hormone is present in the sample.

Minnesota’s newborn screening program has a two-tiered testing approach when screening infants for Congenital Adrenal Hyperplasia. The first test uses a fluoroimmunoasay to screen dried blood spots for a hormone called 17-hydroxyprogesterone (17-OHP). If there is too much 17-OHP in the baby’s dried blood spot, then an additional test will be performed on the blood spot using extracted 17-OHP. If levels remain elevated, the baby may be at risk of having Congenital Adrenal Hyperplasia.

When testing for Congenital Hypothyroidism, the molecule of interest is a hormone called thyroid stimulating hormone (TSH). If the fluoroimmunoassay detects too much TSH in the baby’s dried blood spot, the infant is at risk of having Congenital Hypothyroidism.

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Hemoglobinopathies

Hemoglobinopathies result when there is a problem with the protein (hemoglobin) in the red blood cells that carry oxygen to our tissues and organs. There are several types of hemoglobin. Each type of hemoglobin is abbreviated by using the first letter of its type. For example, hemoglobin F is fetal hemoglobin, hemoglobin A is adult hemoglobin, and hemoglobin S is sickle hemoglobin. People can have more than one type of hemoglobin. Some hemoglobin combinations can cause disease (a hemoglobinopathy). Isoelectric Focusing (IEF) and High Performance Liquid Chromatography (HPLC) are used to identify newborns with a hemoglobinopathy disorder.

Isoelectric focusing is used to separate proteins (hemoglobins in this case). The infant’s sample is placed on one end of a rectangular gel. An electrical field is applied to the gel, which forces the hemoglobins to move through the gel toward either end depending on its electrical charge and the pH of the gel. Different types of hemoglobin travel different distances through the gel. After staining the gel with a colored dye, visible bands can be seen at the location where each hemoglobin stopped traveling. These bands can be compared to known hemoglobins in order to identify the specific hemoglobin(s) present. The most common hemoglobin combination identified in newborn screening is FA, which is a normal result. A baby with an FS result, on the other hand, may be at risk for having Sickle Cell Anemia. Sickle Cell Anemia is one of the most common hemoglobinopathies. A baby identified with the combination, FAS, is not affected with Sickle Cell Anemia but is likely a carrier of the disorder. If the isoelectric focusing test indicates that a baby is at risk for a hemoglobinopathy disorder, High Performance Liquid Chromatography (HPLC) will be used to confirm those results.

High Performance Liquid Chromatography is able to identify the specific type(s) of hemoglobin in the baby’s dried blood spot and how much is present for each type. The infant’s sample is sent through a system of pumps and a column. At the end of the system, it enters a detector where the specific type(s) of hemoglobins are identified based on the amount of time it took them to travel through the column. Information from the detector is then sent to a computer. The computer displays a graph of the detected hemoglobins and how much of each is present. This information is used to determine if a child is at risk for a hemoglobinopathy disorder like Sickle Cell Anemia.

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Galactosemia

Galactosemia results when a specific enzyme (a type of protein) in the body, called galactose-1-phosphate uridyltransferase (GALT), cannot break down galactose (a sugar found in milk). When this enzyme isn’t working correctly, galactose builds up in the body and causes health problems. Screening for Galactosemia involves performing two tests using fluorometry and photometry.

Fluorometry is used to measure the GALT enzyme in the baby’s dried blood spot. This test measures the fluorescence of the sample after a series of chemical reactions. The amount of light released from these reactions can be measured to determine how much GALT enzyme is present. Little or no fluorescence means low or no GALT enzyme activity and the baby is at risk of having Galactosemia.

Photometry is used to assess how well the GALT enzyme is working. If the enzyme is not working correctly, a build-up of galactose will be found in the infant’s dried blood spot. How much galactose is present in the blood is determined by adding a color developing solution to the sample after a series of other chemical reactions. In the presence of galactose, the sample changes color. Upon completion, the absorbance (color) of the sample is read, which is used to calculate the total amount of galactose present. If a baby’s dried blood spot is found to have elevated galactose, then that infant is at risk of having Galactosemia.

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Biotinidase Deficiency

Biotindase Deficiency results when a specific enzyme (biotinidase) is unable to reuse and recycle biotin (one of the B vitamins). Fluorometry is used to measure the biotinidase enzyme in the baby’s dried blood spot. It does this by measuring the amount of light released after a series of chemical reactions. Little or no light means low or no biotinidase enzyme activity and the baby is at risk of having Biotinidase Deficiency.

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Cystic Fibrosis

Minnesota’s Newborn Screening Program has a two-tiered testing approach when screening infants for Cystic Fibrosis (CF). The first test screens dried blood spots for an elevation of an enzyme (a type of protein) called immunoreactive trypsinogen (IRT) by using time-resolved fluoroimmunoassay. Fluoroimmunoassays use two different antibodies (specialized proteins) that bind to a molecule of interest (in this case IRT) present in the baby’s dried blood spot. One of the two antibodies used in the test contains a fluorescent label on it. Fluorescent light is released as a result, which can be measured to determine how much IRT is present. If the fluoroimmunoassay detects elevated levels of IRT, then additional testing on the baby’s dried blood spot is performed. This additional analysis involves performing molecular testing.

Minnesota’s Newborn Screening Program uses molecular testing to look for specific changes (mutations) to the gene responsible for causing Cystic Fibrosis called the Cystic Fibrosis Transmembrane Regulator (CFTR) gene. People with Cystic Fibrosis have mutations in both copies of this gene. There are more than 1,900 mutations known to occur in the CFTR gene and Minnesota’s Newborn Screening Program tests for some of the most common ones. The program tests for 39 mutations and 4 variants that account for ~90% of affected individuals of North American Caucasian background. The detection rate is lower for individuals of other ethnic backgrounds because the mutations known to occur in those ethnicities are rarer.

Minnesota’s Newborn Screening Program tests for the following CF mutations and variants (in italics):

ΔF508 1717-1G>A W1282X 2307insA
ΔI507 R560T N1303K Y1092X>
G542X R553X 394delTT M1101K
G85E G551D Y122X S1255X
R117H 1898+1G>A R347H 3876delA
621+1G>T 2184delA V520F 3905insT
711+1G>T 2789+5G>A A559T  
1078delT 3120+1G>A S549N 5T/7T/9T
R334W R1162X S549R
F508C
R347P 3659delC 1898+5G>T I507V
A455E 3849+10kbC>T 2183AA>G I506V

Genes provide our bodies with the instructions we need to grow and develop. Much like a recipe is used to bake a cake, looking for the specific mutations listed above is like looking at a specific step in the cake recipe to see if it is missing or has misspellings.

If a baby’s dried blood spot is determined to have at least one of the mutations listed above, that infant is at risk of having Cystic Fibrosis.

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Severe Combined Immunodeficiency

Severe Combined Immunodeficiency (SCID) results when there are not enough T-cells (a type of white blood cell that protects the body from infection) in the body. A person with SCID does not have an immune system that works properly, which impacts their ability to fight infection. Screening for SCID involves performing real-time quantitative polymerase chain reaction (qPCR), which allows Minnesota’s Newborn Screening Program to measure the amount of T-cell Receptor Excision Circles (TRECs) in the infant’s dried blood spot. TRECs are small circles of DNA created in our T-cells. If there is little to no TRECs, the baby does not have enough T-cells and is at risk of having SCID or some other primary T-cell lymphopenia (a blood disorder that compromises the immune system), which may include but is not limited to: DiGeorge syndrome, Down syndrome, and CHARGE syndrome.

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Questions

The above descriptions were written as a brief overview of the tests used by Minnesota’s Newborn Screening Program to identify babies at risk for hidden, rare disorders on the newborn screening panel. If a baby is identified as being at risk, newborn screening program staff would contact the baby’s primary care provider and provide them with the information they need to notify the parent(s) and properly follow-up on the result.

For questions or requests for additional information about newborn screening or follow-up methods, please contact the Newborn Screening Program at 800-664-7772.