Newborn Screening Information for Families: Laboratory Testing Overview - Minnesota Dept. of Health

Newborn Screening Information for Families:
Laboratory Testing Overview

Lab testing

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 compounds 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.

We use a test called tandem mass spectrometry (MS/MS) in newborn screening to determine if an infant is at risk for amino acid, fatty acid oxidation, or organic acid disorders. Scientists use the term "mass" to describe a molecule's weight and charge to describe the electrical charge on the molecule. Since every molecule has a unique mass and charge during MS/MS analysis, we are able to identify the specific molecule present based on its mass to charge ratio.

Newborn screening uses a tandem mass spectrometer to identify and measure amino acids and acylcarnitines that 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 tandem mass spectrometer works.

In newborn screening, if too much of an amino acid is present, the infant is at risk for an amino acid disorder. If too many or too few 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, we obtain a DNA analysis to confirm the result. This test looks only for specific changes (mutations) commonly identified in individuals with the disorder. It does not identify all mutations that may be present. 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

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). A time-resolved fluoroimmunoassay (for CAH) and a two site fluoroimmunometric assay (for CH) are used to test for these disorders.

These assays use two different antibodies (specialized proteins) that bind to a molecule of interest (a hormone) present in the blood. One of the two antibodies used in the test contains a fluorescent label on it. Fluorescence 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 CAH. The first test uses the fluoroimmunoassay 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 we obtain an additional test on the blood spot using extracted 17-OHP. If levels remain elevated, the baby may be at risk of having CAH.

When testing for CH, the molecule of interest is a hormone called thyroid stimulating hormone (TSH). If the fluoroimmunometric assay detects too much TSH in the baby's dried blood spot, the infant is at risk of having congenital hypothyroidism.

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.

We use isoelectric focusing to separate proteins (hemoglobins in this case). The infant's sample is placed on one end of a rectangular gel. Then we apply an electrical field 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. We can compare these bands 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 a carrier or at risk for a hemoglobinopathy disorder, we will use high performance liquid chromatography (HPLC) 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.

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.

We measure the GALT enzyme activity in the baby's dried blood spot. This test measures the fluorescence of the sample after a series of chemical reactions simulating digesting galactose. The amount of fluorescence released from these reactions can be measured to determine how much GALT enzyme is working. Little or no fluorescence means low or no GALT enzyme activity and the baby is at risk of having galactosemia.

We also use fluorometry to assess how well the GALT enzyme is working. If the galactose digesting pathway 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 measuring the amount of fluorescently tagged galactose in the sample after a series of other chemical reactions. If a baby's dried blood spot is found to have elevated galactose, that infant is at risk of having galactosemia. Elevated galactose can also suggest an issue with another one of the enzymes in the galactose digesting pathway (epimerase or kinase), especially if the GALT enzyme activity is normal.

Biotinidase deficiency results when a specific enzyme (a type of protein) in the body, called biotinidase (BTD), is unable to free biotin (one of the B vitamins) from the food we eat, so it can be used for energy and growth. We use fluorometry to measure the biotinidase enzyme activity in the baby's dried blood spot.

We measure the BTD enzyme activity in the baby's dried blood spot. This test measures the fluorescence of the sample after a series of chemical reactions simulating the biotin pathway. The amount of fluorescence released from these reactions can be measured to determine how much BTD enzyme is working. Lots of fluorescence means low or no BTD enzyme activity and the baby is at risk of having biotinidase deficiency.

Cystic Fibrosis (CF) is a disorder that affects breathing and digestion (breaking down food). A person with CF makes thick, sticky mucus that blocks the airways of the lungs, making it hard to breathe. This mucus can also make it harder for the body to break down food. The Minnesota 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 immunoreactive trypsinogen (IRT) by using two site fluoroimmunometric assay. Fluoroimmunometric assays 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 fluoroimmunometric assay detects elevated levels of IRT, then we perform additional testing on the baby's dried blood spot. This additional analysis involves performing molecular testing.

We use 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 CF have mutations in both copies of this gene. There are more than 1,700 mutations known to occur in the CFTR gene and our program tests for some of the most common ones. We test for 39 mutations and 4 variants.

In Minnesota, we test 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

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 CF.

Severe combined immunodeficiency (SCID) affects the way the body fights infections. A person with SCID doesn’t have an immune system that works properly. Without a working immune system, the body is unable to fight infections. Screening for SCID involves performing real-time quantitative polymerase chain reaction (qPCR), which allows us 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, which may include but is not limited to: DiGeorge syndrome, Down syndrome, and CHARGE syndrome.

X-linked adrenoleukodystrophy (X-ALD) is a condition that occurs when the body cannot break down certain fats (very long chain fatty acids, or VLCFAs). These fats then build up in the body and affect how the body normally functions. When someone has X-ALD, the buildup of these fats may cause the fatty covering of the nerves, brain, and spinal cord (myelin) to break down. Without myelin, the nerves have a hard time relaying information to the brain. This can cause the nervous system not to function correctly. Symptom severity varies depending on the type of X-ALD and at what age the symptoms start.

We use a test called liquid chromatography tandem mass spectrometry (LC-MS/MS) in newborn screening to determine if an infant is at risk for X-ALD. Scientists use the term "liquid chromatography" to describe how specific compounds are separated, the term "mass" to describe a molecule's weight, and the term "charge" to describe the electrical charge on the molecule. Since every molecule has a unique mass and charge during LC-MS/MS analysis, we are able to identify the specific molecule present based on its mass to charge ratio. Newborn screening uses LC-MS/MS to identify and measure certain lysophosphatidylcholines (LPCs) that are present in a baby's dried blood spot.

In newborn screening, if the baby has high levels of specific LPCs it could mean that the baby is at risk for X-ALD or another disorder such as Zellweger Syndrome.

Lysosomes, the recycling unit of a cell, are sacs that contain enzymes that break down components like sugars and fats into substances the body can use. When lysosomes don’t work properly, it is called a lysosomal disorder (LD). People with lysosomal disorders are not able to break down sugars and fats, which causes them to build-up in the cell. This toxic build up can cause health problems. Lysosomal disorders can affect the brain, liver, spleen, bones, muscle, or heart. There are more than 50 types of lysosomal disorders. The health problems and severity for each depends on the type of lysosomal disorder the person has and at what age the health problems began. To screen for LDs, we use a test called FIA-MS/MS (Flow Injection Analysis Tandem Mass Spectrometry). FIA-MS/MS is an instrument that measures the rate at which the enzymes break down components. If a baby has lower than normal rates, it could mean that the baby is at risk for a lysosomal disorder.

Flow injection analysis means that all the sample preparation is done before putting the samples on the instrument. Tandem mass spectrometry is a series of three chambers in the instrument that work together to measure the amount of specific molecules in a sample. In the explanation below, we use a coin sorter as an example to explain how this process works.

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 many of the target molecules 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.

The above descriptions are designed to give a brief overview of the tests we use in Minnesota to identify babies at risk for hidden, rare disorders on the newborn screening panel. If a baby is identified as being at risk, our program staff contacts a member of the baby's healthcare team and provides 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, contact us at 800-664-7772.