The first analyses of blood spot samples from the National Institutes of Health's National Children's Study are now underway in the laboratories of two members of AACC's Pediatric Reference Range Committee (PRRC), Patti Jones, PhD, and Dennis Dietzen, PhD. Using tandem mass spectrometry, the researchers are conducting studies of the precious samples collected from healthy newborns to define age-specific reference ranges for steroid hormones and amino acids that impact children's development.

"It took a decade for the National Children's Study to get to where it is now," said Michael Bennett, PhD, FRCPath, referring to the efforts that are aimed at enlisting a diverse population of 100,000 healthy children at birth and following them for 21 years. As the study kicked off, a contingent of AACC members sought to collaborate with the NIH researchers in order to access blood samples from the participants. Bennett chairs the PRRC and is director of the Michael J. Palmieri Metabolic Laboratory at Children's Hospital of Philadelphia and professor of pathology and laboratory medicine at the University of Pennsylvania Perelman School of Medicine.

At a press conference held during the AACC Annual Meeting in Houston, Jones and Dietzen described early results from their research. Jones, who is clinical director of the chemistry and metabolic disease labs at Children's Medical Center of Dallas and professor of pathology and medical laboratory science at UT Southwestern Medical Center, is leading the arm of the study focused on steroid hormones, including 17-OH progesterone, dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androstenedione, and testosterone.

"The reason we picked these steroids is that they are the tests that are ordered to diagnose and monitor congenital adrenal hyperplasia. This disorder in kids can have a serious effect on the development of their primary and secondary sexual characteristics. In addition, lack of production of some steroids in this pathway are involved in a very serious illness called salt wasting, where the body can't regulate and maintain the amount of sodium," Jones explained. "Having well-defined reference ranges is therefore important to interpreting these tests."

In the first studies of 17-OH progesterone, Jones found concentrations in newborns span a large range: 5 – >1,000 ng/dL. An early observation is that there are some differences between male and female newborns, but Jones was particularly curious about the samples from the high end of the range. It turns out that these were from babies less than 24 hours old; however, by 48 hours, the levels of this steroid hormone drop down to the low end of the range. If this observation holds up, it would mean that babies with levels at the high end of the range who are >48 hours of age would be abnormal and require medical interventions, Jones said. "That's why this study is so near and dear to my heart. I want to be able to have those ranges to interpret test results." So far, she has analyzed about 300 samples by tandem mass spectrometry and will continue studies to analyze the other targeted steroid hormones.

Dietzen is analyzing the same newborn blood spots for amino acids. "I like to think of amino acids as the canary in the coal mine because they are key components of multiple metabolic processes," he said. In addition to protein synthesis, amino acids play a role in providing energy, participate in signaling systems involved in blood pressure regulation, and serve as neurotransmitters in the brain. Using a tandem mass spectrometry method developed in his lab, Dietzen can simultaneously analyze and quantitate 32 amino acids from a single sample in 15–20 minutes. While some of the 32 are analyzed in newborn screening programs, many are not.

In the 160 samples analyzed so far, Dietzen rejected two that showed evidence of inborn errors of metabolism. He also found that six amino acids normally picked up by the analysis—alloisoleucine, homocystine, cystathionine, cystine, argininosuccinate, and alpha-aminoadipic acid—are undetectable in the spots as their concentrations are too low. In time and with more samples, Dietzen aims to develop ranges for 26 amino acids.

Looking toward the future, Dietzen is hopeful that following study participants will provide not only a picture of group variability, but also individual variability that can be related to genetic and environmental factors.