NEW YORK – Researchers from the US Centers for Disease Control and Prevention have developed a method to test newborns for homocystinuria, a condition that can lead to permanent damage or death and is often missed by common newborn screening assays.
In describing the test in a study published last week in Clinical Chemistry, the authors noted that homocystinuria affects an infant's ability to metabolize methionine and leads to an increase in levels of both methionine and another biomarker, homocysteine. Further, it can cause eye and skeletal issues, intellectual disabilities, and vascular abnormalities.
Traditionally, screening for the disease involves using multiplex fast flow injection analysis mass spectrometry (FIA-MS/MS) tests that use methionine as a biomarker, which is often still low at the time newborns are screened. The use of methionine as a biomarker "has led to newborns with homocystinuria being missed in the past," said Konstantinos Petritis, laboratory chief of the biochemical mass spectrometry lab in the newborn screening and molecular biology branch of the CDC and one of the developers of the test.
Also, in current newborn screening for homocystinuria, a sample of a newborn's dried blood spot is taken, mixed with isotopically labeled internal standards and solvents, and analyzed by FIA-MS/MS. Because dozens of diseases are being screened in the same newborn, that can interfere with homocystinuria biomarker measurements, Petritis said.
With the new CDC test, the researchers introduced a reduction step that releases bound oxidized homocysteine, followed by a derivatization step that increases the sensitivity for homocysteine. Petritis noted that both the molecular weight and fragmentation pattern after the derivatization step are unique to homocysteine, which eliminates the effects of interference from the compounds and internal standards that are used for measuring other biomarkers. The additional steps add 20 minutes to the sample preparation time, and the limits of detection were comparable to current methods, the researchers noted in Clinical Chemistry.
According to Petritis, this test is the first that allows homocysteine to be multiplexed in a commonly used FIA-MS/MS newborn screening assay. Although there are other tests for total homocysteine that use separation before analysis by mass spectrometry — which are currently used as second-tier screening tests for infants with high methionine levels — those tests have longer analysis times and aren't as heavily multiplexed as the FIA-MS/MS assay, he said.
Only using homocysteine as a second-tier screening biomarker can miss babies that have homocystinuria but have low methionine levels, since only those with higher methionine levels are sent for second-tier screening, he said. Because newborn screening samples are collected within the first two days of an infant's life, the methionine levels may not yet be high enough to indicate a need for further screening.
Methionine "has the inherent problem of lower clinical sensitivity for homocystinuria," Petritis said, and it takes longer to "elevate to the decision point where a newborn would be classified as presumptive positive for homocystinuria."
To reduce the risk of false negatives, laboratories can lower methionine cutoffs, but that results in lower specificity, the researchers wrote in the Clinical Chemistry paper. To mitigate that decline in specificity, they can use a methionine-to-phenylalanine ratio or perform second-tier screening for homocystinuria, but those options can cause higher false positive rates and lower throughput or require additional equipment and personnel needs, they said.
In addition, Petritis said, methionine levels are affected by the total parenteral nutrition solution (TPN) that is given to premature or sick newborns in neonatal intensive care units. Homocysteine, meantime, "is closer metabolically to homocystinuria, it elevates earlier in affected newborns, and is not influenced by the administration of TPN."
The research team tested its method on 152 residual clinical specimens to validate the assay's ability to distinguish 100 presumptive normal samples from babies who had not received TPN, 50 samples from infants who had received TPN, and two samples from babies with confirmed homocystinuria. Their method was able to accurately classify positive samples while distinguishing the positive samples from those with TPN administration, the researchers wrote.
Use of the test is not limited to the CDC's laboratory. The additional chemicals to analyze homocysteine were developed and optimized to allow for "seamless integration" into both existing and future first-tier newborn screening assays, making it possible for any lab to implement, he said. Those chemicals are also widely available at a reasonable cost.
Newborn screening laboratories or kit manufacturers can either try to modify their existing assays using the CDC's approach or adopt the test as is, Petritis said, and the researchers have been in contact with laboratories and manufacturers that may be interested in piloting or adopting the test.
The next step for moving the test toward clinical implementation is for a laboratory to pilot the assay and offer feedback on its performance after using it on thousands of specimens. According to Petritis, there "has been some traction towards that goal recently."
Mei Baker, director of the newborn screening laboratory at the Wisconsin State Laboratory of Hygiene, said that there is "no question" that homocysteine is a much better marker than methionine in newborn screening for homocystinuria. Baker said that she was confident the method can be adopted in public newborn screening labs like hers and that to her knowledge, it is the only reported homocysteine measurement method that can be multiplexed with other mass spectrometry analytes used in newborn screening.
She added that the test needs to be further evaluated in a routine newborn screening setting and undergo regulatory compliance validation. Baker also noted that there are other genetic defects that may cause elevated homocysteine, which are beneficial to identify during newborn screening.
Detecting other diseases related to homocysteine levels was raised as an option by Petritis as well. The ability to add homocysteine analysis into primary tandem mass spectrometry screening "opens the door for the detection of other rare metabolic diseases that use total homocysteine as a biomarker," Petritis said, naming re-methylation disorders as examples. The research team is also working to multiplex two more biomarkers into the test that will be able to differentiate homocystinuria from other diseases, he said.