Dana Bailey

Analytical measurement and clinical relevance of vitamin D3 C3-epimer

With an ever-increasing clinical interest in vitamin D insufficiency, numerous automated immunoassays, protein binding assays, and in-house LC-MS/MS methods are being developed for the quantification of 25-hydroxyvitamin D(3) (25(OH)D(3)). Recently, LC-MS/MS methods have identified an epimeric form of 25(OH)D(3) that has been shown to contribute significantly to 25(OH)D(3) concentration, particularly in infant populations. This review describes the metabolic pathway and physiological functions of 3-epi-vitamin D, compares the capability of various 25(OH)D(3) methods to detect the epimer, and highlights recent publications quantifying 3-epi-25(OH)D(3) in infant, pediatric, and adult populations. In total, this review summarizes the information necessary for clinicians and laboratorians to decide whether or not to report/consider the C3-epimer in the analysis and clinical assessment of vitamin D status.

Marked Biological Variance in Endocrine and Biochemical Markers in Childhood: Establishment of Pediatric Reference Intervals Using Healthy Community Children from the CALIPER Cohort

BACKGROUND Reference intervals are indispensable in evaluating laboratory test results; however, appropriately partitioned pediatric reference values are not readily available. The Canadian Laboratory Initiative for Pediatric Reference Intervals (CALIPER) program is aimed at establishing the influence of age, sex, ethnicity, and body mass index on biochemical markers and developing a comprehensive database of pediatric reference intervals using an a posteriori approach. METHODS A total of 1482 samples were collected from ethnically diverse healthy children ages 2 days to 18 years and analyzed on the Abbott ARCHITECT i2000. Following the CLSI C28-A3 guidelines, age- and sex-specific partitioning was determined for each analyte. Nonparametric and robust methods were used to establish the 2.5th and 97.5th percentiles for the reference intervals as well as the 90% CIs. RESULTS New pediatric reference intervals were generated for 14 biomarkers, including α-fetoprotein, cobalamin (vitamin B12), folate, homocysteine, ferritin, cortisol, troponin I, 25(OH)-vitamin D [25(OH)D], intact parathyroid hormone (iPTH), thyroid-stimulating hormone, total thyroxine (TT4), total triiodothyronine (TT3), free thyroxine (FT4), and free triiodothyronine. The influence of ethnicity on reference values was also examined, and statistically significant differences were found between ethnic groups for FT4, TT3, TT4, cobalamin, ferritin, iPTH, and 25(OH)D. CONCLUSIONS This study establishes comprehensive pediatric reference intervals for several common endocrine and immunochemical biomarkers obtained in a large cohort of healthy children. The new database will be of global benefit, ensuring appropriate interpretation of pediatric disease biomarkers, but will need further validation for specific immunoassay platforms and in local populations as recommended by the CLSI.

Complex Biological Pattern of Fertility Hormones in Children and Adolescents: A Study of Healthy Children from the CALIPER Cohort and Establishment of Pediatric Reference Intervals

BACKGROUND Pediatric endocrinopathies are commonly diagnosed and monitored by measuring hormones of the hypothalamic-pituitary-gonadal axis. Because growth and development can markedly influence normal circulating concentrations of fertility hormones, accurate reference intervals established on the basis of a healthy, nonhospitalized pediatric population and that reflect age-, gender-, and pubertal stage-specific changes are essential for test result interpretation. METHODS Healthy children and adolescents (n = 1234) were recruited from a multiethnic population as part of the CALIPER study. After written informed parental consent was obtained, participants filled out a questionnaire including demographic and pubertal development information (assessed by self-reported Tanner stage) and provided a blood sample. We measured 7 fertility hormones including estradiol, testosterone (second generation), progesterone, sex hormone-binding globulin, prolactin, follicle-stimulating hormone, and luteinizing hormone by use of the Abbott Architect i2000 analyzer. We then used these data to calculate age-, gender-, and Tanner stage-specific reference intervals according to Clinical Laboratory Standards Institute C28-A3 guidelines. RESULTS We observed a complex pattern of change in each analyte concentration from the neonatal period to adolescence. Consequently, many age and sex partitions were required to cover the changes in most fertility hormones over this period. An exception to this was prolactin, for which no sex partition and only 3 age partitions were necessary. CONCLUSIONS This comprehensive database of pediatric reference intervals for fertility hormones will be of global benefit and should lead to improved diagnosis of pediatric endocrinopathies. The new database will need to be validated in local populations and for other immunoassay platforms as recommended by the Clinical Laboratory Standards Institute.

Maternal–fetal–infant dynamics of the C3-epimer of 25-hydroxyvitamin D

BACKGROUND Poor vitamin D status (i.e. low serum 25-hydroxyvitamin D (25(OH)D)) has been associated with adverse clinical outcomes during pregnancy and childhood. However, the interpretation of serum 25(OH)D levels may be complicated by the presence of the C3-epimer of 25(OH)D. We aimed to quantify C3-epi-25(OH)D3 in pregnant women and fetuses, to explore the relationship of the C3-epimer between maternal and cord samples, and to establish whether infant C3-epimer abundance is explained by prenatal formation. METHODS In a sub-study of a randomized trial of prenatal vitamin D3, 25(OH)D3 and C3-epi-25(OH)D3 were quantified by LC-MS/MS in 71 sets of mother-fetus-infant serum samples, including maternal delivery specimens, cord blood, and infant specimens acquired at 3-28 weeks of age. RESULTS Without supplementation, median concentrations of C3-epi-25(OH)D₃ were higher in infants (6.80 nmol/L) than mothers (0.45 nmol/L) and cord blood (0 nmol/L). However, there was substantial variation such that C3-epi-25(OH)D₃ accounted for up to 11% (maternal), 14% (cord), and 25% (infant) of the total 25(OH)D₃. Supplemental vitamin D₃ significantly increased maternal-fetal C3-epi-25(OH)D₃, and was a preferential source of C3-epi-25(OH)D₃ compared to basal vitamin D, possibly due to C3-epi-cholecalciferol in the supplement. Multivariate regression did not suggest transplacental transfer of C3-epi-25(OH)D₃, but rather indicated its generation within the fetal-placental unit from maternally-derived 25(OH)D₃. Neither maternal nor fetal C3-epi-25(OH)D₃ is accounted for the relatively high concentrations of infant C3-epi-25(OH)D₃, suggesting rapid postnatal generation. CONCLUSIONS C3-epi-25(OH)D₃ is present in some pregnant women and fetuses, but does not appear to be efficiently transferred transplacentally. High C3-epimer concentrations in infancy are probably due to postnatal formation rather than fetal stores.

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.

Pediatric Within-Day Biological Variation and Quality Specifications for 38 Biochemical Markers in the CALIPER Cohort

BACKGROUND Studies of biological variation provide insight into the physiological changes that occur within and between study participants. Values obtained from such investigations are important for patient monitoring and for establishing quality specifications. In this study we evaluated the short-term biological variation of 38 chemistry, lipid, enzyme, and protein analytes in a pediatric population, assessed the effect of age partitions on interindividual variation, and compared the findings to adult values. METHODS Four plasma samples each were obtained within 8 h from 29 healthy children (45% males), age 4-18 years. Samples were stored at -80 °C and analyzed in 3 batches, with samples from 9-10 study participants per batch. Within-person and between-person biological variation values were established using nested ANOVA after exclusion of outliers by use of the Tukey outlier test. Analytical quality specifications were established with the Fraser method. RESULTS Biological variation coefficients and analytical goals were established for 38 analytes. Age partitioning was required for 6 analytes. Biological variation characteristics of 14 assays (37%) were distinct from adult values found in the Westgard database on biological variation. Biological variation characteristics were established for 2 previously unreported analytes, unconjugated bilirubin and soluble transferrin receptor. CONCLUSIONS This study is the first to examine biological variation and to establish analytical quality specifications on the basis of biological variation for common assays in a pediatric population. These results provide insight into pediatric physiology, are of use for reference change value calculations, clarify the appropriateness of reference interval use, and aid in the development of quality management strategies specific to pediatric laboratories.

Use of MALDI-TOF for Diagnosis of Microbial Infections

Although mass spectrometry is making its mark on all facets of clinical laboratory medicine, arguably no field is witnessing its impact more than clinical microbiology. The application of MALDI-TOF mass spectrometry (MALDI-TOF MS) to microbial identification is revolutionizing clinical microbiology by providing rapid identification with minimal sample preparation at a potential savings in costs. Across the globe, the degree of implementation of MALDI-TOF MS varies markedly. In Canada, Australia, and much of Europe, MALDI platforms are in routine use in clinical microbiology, whereas the US Food and Drug Administration has yet to provide clinical clearance. In this Q&A, 4 experts from across the globe with first-hand experience implementing MALDI-TOF MS in the microbiology laboratory provide insight into what this technology can and cannot provide, what it takes to bring it in house, and what direction it takes us in the future. The application of MALDI-TOF MS to the diagnosis of microbial infections has been touted as a revolution in clinical microbiology. However, no technology is without its pitfalls. Can you please describe what you feel are the greatest strengths and limitations of MALDI-TOF MS? Gilbert Greub: When it is used to identify bacterial strains and fungi, the main strengths of MALDI-TOF MS are the rapidity of the technique ( 95% accuracy at the species level. One of the most important limitations of this technique is its relatively low analytical sensitivity (about 105–106 bacteria/well). Thus, the accuracy of the identification is increased when the identification is done on a colony grown on agar or on a blood culture pellet, i.e., after a culture-based amplification step. Consequently, MALDI-TOF MS is not a tool currently suitable to detect …

A next generation enzymatic magnesium assay on the Abbott ARCHITECT chemistry system meets performance goals based on biological variation

OBJECTIVE To evaluate the performance of the Abbott ARCHITECT enzymatic assay for magnesium (3P68) in serum/plasma and urine against analytical goals based on biological variation. METHODS Analytical performance was evaluated according to CLSI protocols. Precision was examined using commercial chemistry controls. Accuracy was assessed against NIST SRM 956c, electrolytes in human serum. Correlation with the arsenazo Mg assay (7D70) was completed using patient samples (plasma, N = 101; urine, N = 90). Common interferences were examined in pooled patient specimens with high and low magnesium concentrations. RESULTS The enzymatic Mg assay displayed imprecision of 1.7% at 0.72 mmol/L and 1.4% at 1.80 mmol/L (20 days, one calibration, one reagent lot). The linear range was verified between 0.18-7.0 mmol/L (plasma) and 0.01-10.69 mmol/L (urine). Results of the enzymatic assay (x) correlated well with the predicate assay (y) with the relationships y = 0.891x + 0.035, R = 0.967 (plasma) and y = 1.181x + 0.086, R = 0.997 (urine). Mean bias of the NIST SRM 956 c samples was -1.4%. This method showed minimal interference by hemoglobin (3g/L as hemolysate), lipemia (20 g/L Intralipid), unconjugated bilirubin (531 μmol/L), and ascorbate (680 μmol/L). CONCLUSIONS The ARCHITECT Magnesium assay 3P68 achieved the desirable analytical quality specification of 4.8% for total allowable error. In comparison to the 7D70 assay, notable improvements are seen in precision, 30-day calibration stability, and minimal interference by hemolyzed and lipemic samples.