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Seattle Children's Developing Mass Spec Tests for Newborn Screening


NEW YORK (360Dx) – Scientists at Seattle Children's Hospital are developing mass spectrometry-based protein tests for screening newborns for various immunodeficiency disorders.

The researchers have formed a company, Key Proteo, to support the effort and are planning a large-scale pilot study to evaluate their assays, said Sihoun Hahn, principal investigator at Seattle Children's Research Institute and leader of the effort.

Hahn has received four grants from the National Institute of Allergy and Infectious Diseases totaling $3.7 million in support of the work.

He and his colleagues are using immuno-selected-reaction monitoring (SRM) mass spec to measure proteins of interest in dried blood spots. SRM mass spec analysis of dried blood spot samples is commonly used in newborn screening applications, but these tests usually target small molecules, as opposed to peptides or proteins. By expanding testing to certain proteins, the researchers hope to enable detection of immunodeficiency disorders that are not currently tested for in typical newborn screening programs.

Hahn came to focus on the development of immuno-SRM screening tests through his interest in Wilson's Disease, a disorder in which the body is not able to properly transport copper, leading to its accumulation in various organs causing neurological and liver damage. The outcomes can be avoided by early detection and intervention, which make it a promising target for newborn screening assays.

The disease is caused by alterations in the ATP7B gene that cause patients to express either very low level or no ATP7B protein, which, Hahn said, opened up the possibility of screening for the disorder by testing newborns' ATP7B levels.

Because newborn screening samples are typically collected as dried blood spots and analyzed using mass spectrometry, the ATP7B test would ideally also use mass spec to measure the protein in those same dried blood spot samples.

Hahn and his colleagues initially tried to measure the protein in dried blood spots without any upfront enrichment but found that their mass spec assay wasn't sensitive enough. This led them to try the immuno-SRM approach, which uses antibodies to enrich target peptides prior to mass spec analysis, which improves the sensitivity of the assay. To help develop the assay, Hahn collaborated with Fred Hutchinson Cancer Research Center researchers Amanda Paulovich and Jeffrey Whiteaker, both experts in immune-SRM assay development.

In a 2016 study in the Journal of Proteome Research, the researchers presented data showing that an immuno-SRM assay to ATP7B could quantify the protein in dried blood spots at picomolar concentrations and with coefficients of variation of under 10 percent. They measured levels of the protein in 12 healthy controls and 13 patients with Wilson's Disease, finding that 12 of the 13 patients with Wilson's had ATP7B levels well below the control range, suggesting, they wrote, "the feasibility of the use of ATP7B as a potential marker for" the condition.

With those results providing a proof-of-concept, Hahn and his colleagues expanded their work to look at other conditions that might be detected using immuno-SRM as part of a newborn screening program. In a study published last month in Frontiers in Immunology, the researchers tested a multiplexed assay for detecting X-linked agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), and CD3ϵ-associated severe combined immunodeficiency (SCID) by measuring, respectively, the proteins WASp, BTK, and CD3ϵ in dried blood spots. Testing 42 patients with one of these conditions along with 40 healthy adult controls and 62 healthy newborns, they found that in 97.6 percent of cases the results of the immuno-SRM assays matched the patient's clinical diagnosis.

Based on this work, Hahn and his colleagues are now looking to run a large pilot trial testing their assays in around 50,000 to 100,000 newborn screening samples.

"That will let us determine the detection rates, the false positive and false negative rates," he said, adding that if the assays show strong enough performance in the pilot trial the next step would be to promote the tests for nomination by the US Department of Health and Human Services Advisory Committee on Heritable Disorders in Newborns and Children, which recommends tests for inclusion in state newborn screening programs.

Hahn said that he launched Key Proteo to help raise the funds and resources required for the pilot study. The company is currently working with a consultant to raise funding and establish a business plan and organizational structure, he said.

He said that he and his collaborators plan to evaluate assays for three to four conditions in the pilot study but that they are developing tests for 12 to 15 different diseases with the goal ultimately of building a multiplexed assay for all of them. He said he believed the assay could be run at a cost of around $5 per sample.

The researchers plan to package the assays as an in vitro diagnostic kit that can be run by state newborn screening laboratories. If successful, the effort would represent a significant step for clinical mass spec, demonstrating not only that immuno-SRM assays could effectively measure proteins in dried blood spot samples in a clinical environment, but also that these assays could be packaged as kits and run reproducibly across different laboratories.

Hahn acknowledged the challenges involved in this undertaking but said that he and his colleagues "have made a lot of progress in terms of the assay reproducibility and stability."

He said that they were currently working to build a platform capable of running the large number of samples they will target in the pilot study, which they hope to start within one to two years.

While dried blood spots have been used for years in newborn screening, they have drawn growing interest from other areas of clinical mass spec in recent years due to their potential to enable easier patient sampling and lower assay costs.

Because dried blood spots use a finger prick as opposed to conventional phlebotomy, patients could conceivably draw samples themselves without needing to visit a doctor's office, which could open up a number of applications around, among other things, longitudinal monitoring of chronic conditions or even basic wellness monitoring.

Additionally, dried blood spots could significantly reduce sample shipping costs as they don't require the rapid cold shipping commonly used for conventional blood samples.

One issue that has limited the uptake of dried blood spots for protein testing is the fact that traditional immunoassays are poorly suited to analyzing these samples given their small volume. The rise of mass spec as a clinical proteomics tool, however, has made them a plausible sample source for protein-based tests.

One major proponent of dried blood spot protein assays is SISCAPA Assay Technologies CEO Leigh Anderson, whose SISCAPA technology is a variation of the immuno-SRM approach used by Hahn's lab.

Anderson, who is not involved in Hahn's work, said that the research "looks very persuasive and is something that makes a lot of sense logistically. It's one more demonstration that you can do useful protein measurements in dried blood spots."

He noted that newborn screening also made sense as an area where immuno-SRM testing could make inroads given that one would expect to see significant differences in the expression of target proteins between cases and controls due to the nature of the diseases being investigated.

"They are basically screening for genetic diseases, so I'm not sure how quantitative the assays need to be," Anderson said. "The constraints [around quantitative precision] might be less for implementing those kinds of assays."

He noted, though, that moving the tests through the US Food and Drug Administration as is required for IVD kits could prove challenging.

"The cost of doing the studies to get FDA clearance for kits like that is significant, and one of the issues with neonatal screening is that it is not a big-dollar business," he said. "That has been a barrier to the conversion of these mass spec protein tests over to FDA-cleared in vitro diagnostic kits."