Dynamic biological changes in metabolic disease biomarkers in childhood and adolescence: A CALIPER study of healthy community children.

BACKGROUND: Understanding age- and sex-specific biological changes in metabolic disease biomarkers is essential for their appropriate utilization in management of children with inborn errors of metabolism (IEM). The CALIPER program aimed to establish pediatric reference values in healthy community children for common metabolic biomarkers and determine the effects of key covariates including age and sex across the pediatric age. METHODS: A cohort of 500 healthy children and adolescents from birth to 19years were initially recruited to establish pediatric reference intervals according to the CLSI C28-A3 guidelines. Serum samples were used to measure 37 amino acids by ultra-performance liquid chromatography, 32 acylcarnitines, as well as free and total carnitine by tandem mass spectrometry, and ß-hydroxybutyrate and free fatty acids using the Vitros 5.1 chemistry analyzer. P ediatric reference intervals were calculated using non-parametric statistics and partitioned based on age- and sex-distributions. RESULTS: Approximately 80% of all analytes required 2 to 4 age-dependent partitions, with over 50% of amino acids and over 70% of acylcarnitines exhibiting significant physiological changes during the neonatal period. Also, 21% of all analytes required partitioning during puberty and adolescence, half of which produced sex-specific distributions. CONCLUSIONS: A comprehensive reference interval database for metabolic disease biomarkers established in this study will improve detection of IEMs by providing appropriate age- and sex-related information in the pediatric population. It will also aid newborn screening programs and guide the management of patients with known metabolic diseases, especially pubertal and adolescent boys and girls that display sex-specific concentrations.

Pediatric reference value distributions for vitamins A and E in the CALIPER cohort and establishment of age-stratified reference intervals.

OBJECTIVES: Vitamin A (retinol) and vitamin E (α-tocopherol) are fat soluble micronutrients most commonly measured in the pediatric population to monitor deficiencies due to malabsorption secondary to gastrointestinal (GI) disorders. One of the major challenges of vitamin A and E testing is the lack of reliable pediatric reference intervals which limits accurate interpretation of results. Here we report new pediatric reference intervals (RI) for both vitamins as part of the Canadian Laboratory Initiative for Pediatric Reference Intervals (CALIPER). DESIGN AND METHODS: A total of 342 blood samples were collected from healthy children 1 day to 19 years of age recruited from the community. Retinol and α-tocopherol were extracted from serum using hexane before concentrations were measured with high-performance liquid chromatography (HPLC). Age and sex-specific RI were calculated with guidance from CLSI C28-A2. Non-parametric and robust methods were used to calculate the 95th percentile ranges of the reference intervals along with the 90% confidence intervals. RESULTS: Vitamin A demonstrated increasing levels with age necessitating four distinct age stratifications. Vitamin E levels peaked within the first year of life requiring only 2 age partitions. Ratios of vitamin E to cholesterol and triglyceride were also calculated, and correlated well to vitamin E levels. Sex-specific differences were not observed. CONCLUSIONS: This study establishes pediatric RI for vitamins A and E in a healthy population of children from neonates to early adulthood. These values will be beneficial in assessing accurate vitamin status when monitoring children with GI disorders or malnutrition.

Analytical measurement of serum 25-OH-vitamin D3, 25-OH-vitamin D2 and their C3-epimers by LC-MS/MS in infant and pediatric specimens.

OBJECTIVES: To develop a simple and sensitive LC-MS/MS procedure for quantification of serum 25-OH-vitamin D3 (25-OH-D3), 25-OH-vitamin D2 (25-OH-D2), and their C3-epimers. METHODS: Serum 25-OH-vitamin D metabolites were extracted with MTBE and quantified by LC-MS/MS. Commercially available calibrators and QC materials were employed. The ion-transition 401.2→365.2 was monitored for 25-OH-D3 and C3-epi-25-OH-D3, 407.2→371.3 for d6-25-OH-D3, 413.2→331.2 for 25-OH-D2 and C3-epi-25-OH-D2 and 419.2→337.1 for, d6-25-OH-D2. As a proof-of-principle, 25-OH-D3 and C3-epi-25-OH-D3 were quantified in 200 pediatric subjects (0-20 years of age). Cholecalciferol supplements were examined as a potential source of C3-epimer. RESULTS: The assay provided an LLOQ of ≤2.8 nmol/L for all 25-OH-D metabolites, with a linear response up to 400 nmol/L. The CV was <10% for 25-OH-D2/3 and <15% for C3-epi-25-OH-D3. C3-epi-25-OH-D3 was quantified in all subjects, with higher concentrations observed in infants ≤1 year of age (11.44 nmol/L vs. 4.4 nmol/L; p<0.001). Within the first year of life, 25-OH-D3 concentrations increased linearly, while C3-epi-25-OH-D3 concentrations remained constant. At 12 months of age, C3-epi-25-OH-D3 concentration dropped by almost 50% (11.4 nmol/L in infants ≤1year of age vs. 5.4 nmol/L in infants 1-2years of age; p<0.001). Liquid vitamin D3 supplements did not contain appreciable amounts of C3-epi-D3. CONCLUSIONS: The proposed LC-MS/MS procedure is suitable for quantifying 25-OH-D3 metabolites. Although the C3-epimer is present in all pediatric subjects, it is significantly elevated in individuals ≤1 year of age and drops at 12 months of age. Oral vitamin D supplements are unlikely to be a significant source of C3-vitamin D epimer.

Analytical performance and workflow evaluation of the Roche E170 modular immunoassay analyzer in a pediatric setting.

OBJECTIVES: To evaluate the analytical performance of the Roche E170 modular analytics immunoassay analyzer and assess its impact on workflow efficiency and ability to consolidate workstations in a pediatric setting. DESIGN AND METHODS: Analytical performance of eleven common immunoassays was assessed. Total imprecision was determined using Roche PreciControl Universal controls, Bio-Rad Lyphochek Immuno Plus, Anemia controls, and a human serum pool. Method comparison was performed with approximately 100 patient specimens. High dose hook effect, sample carryover, and results comparison between the two measuring channels were evaluated. For the workflow study, the time required for sample and reagent handling, instrument preparation, and hands-on time were assessed. RESULTS: Correlation coefficients with existing methods ranged from 0.941 to 0.999. Biases of -19% to 70% were observed. Total imprecision ranged from 1.1 to 7.6%. No sample carryovers were encountered. Results from both measuring channels were comparable. CONCLUSION: E170 is suitable for use in a pediatric setting. The analytical performance is acceptable and gives equivalent results to our existing systems. The precision is comparable and acceptable. Some improvement in efficiency, workflow, cost saving, and consolidation of workstations is possible. Significant workflow improvements can only be realized when integrated with the chemistry modules.