Man Khun Chan

Closing the Gaps in Pediatric Laboratory Reference Intervals: A CALIPER Database of 40 Biochemical Markers in a Healthy and Multiethnic Population of Children

BACKGROUND Pediatric healthcare is critically dependent on the availability of accurate and precise laboratory biomarkers of pediatric disease, and on the availability of reference intervals to allow appropriate clinical interpretation. The development and growth of children profoundly influence normal circulating concentrations of biochemical markers and thus the respective reference intervals. There are currently substantial gaps in our knowledge of the influences of age, sex, and ethnicity on reference intervals. We report a comprehensive covariate-stratified reference interval database established from a healthy, nonhospitalized, and multiethnic pediatric population. METHODS Healthy children and adolescents (n = 2188, newborn to 18 years of age) were recruited from a multiethnic population with informed parental consent and were assessed from completed questionnaires and according to defined exclusion criteria. Whole-blood samples were collected for establishing age- and sex-stratified reference intervals for 40 serum biochemical markers (serum chemistry, enzymes, lipids, proteins) on the Abbott ARCHITECT c8000 analyzer. RESULTS Reference intervals were generated according to CLSI C28-A3 statistical guidelines. Caucasians, East Asians, and South Asian participants were evaluated with respect to the influence of ethnicity, and statistically significant differences were observed for 7 specific biomarkers. CONCLUSIONS The establishment of a new comprehensive database of pediatric reference intervals is part of the Canadian Laboratory Initiative in Pediatric Reference Intervals (CALIPER). It should assist laboratorians and pediatricians in interpreting test results more accurately and thereby lead to improved diagnosis of childhood diseases and reduced patient risk. The database will also be of global benefit once reference intervals are validated in transference studies with other analytical platforms and local populations, as recommended by the CLSI.

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.

Canadian Laboratory Initiative on Pediatric Reference Interval Database (CALIPER): Pediatric reference intervals for an integrated clinical chemistry and immunoassay analyzer, Abbott ARCHITECT ci8200 ☆ ☆☆

OBJECTIVES A comprehensive set of age- and gender-specific pediatric reference intervals is essential for accurate interpretation of laboratory tests in a pediatric setting. DESIGN AND METHODS 1459 serum/plasma from children attending select outpatient clinics and deemed to be metabolically stable, were collected from five age groups; 0-12 months, 1-5 years, 6-10 years, 11-14 years and 15-20 years. Samples were analyzed for 24 chemistries and 15 immunoassays on ARCHITECT ci8200. RESULTS Reference intervals were established according to CLSI/IFCC C28-P3 guidelines by the Robust statistical method. The ranges reflect the central 95% confidence intervals for the population tested. Age and gender were partitioned using the Harris-Boyd method. CONCLUSIONS While these intervals are ci8200 method specific, they not only provide robust intervals for users of this system but are also useful for any laboratory requiring pediatric intervals if they can be shown to be transferable and if validated for the local patient population.

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.

A sensitive and rapid mass spectrometric method for the simultaneous measurement of eight steroid hormones and CALIPER pediatric reference intervals

OBJECTIVES To develop an accurate assay and establish the normal reference intervals for serum cortisol, corticosterone, 11-deoxycortisol, androstenedione, 21-hydroxyprogesterone, testosterone, 17-hydroxyprogesterone, and progesterone. These steroids are commonly used as biomarkers for the diagnosis and monitoring of endocrine diseases such as congenital adrenal hyperplasia. Appropriate age- and gender-stratified reference intervals are essential in accurate interpretation of steroid hormone levels. DESIGN AND METHODS The samples analyzed in this study were collected from healthy, ethnically diverse children in the Greater Toronto Area as part of the CALIPER program. A total of 337 serum samples from children between the ages of 0 and 18years were analyzed. The concentrations were measured by using an LC-MS/MS method. The data were analyzed for outliers and age- and gender-specific partitions were established prior to establishing the 2.5th and 97.5th percentiles for the reference intervals. RESULTS Reference intervals for all hormones required significant age-dependent stratification while testosterone and progesterone required additional sex-dependent stratification. CONCLUSIONS We report a sensitive, accurate and relatively fast LC-MS/MS method for the simultaneous measurement of eight steroid hormones. Detailed reference intervals partitioned based on both age and gender were also established for all eight steroid hormones.

Pediatric reference intervals for 28 chemistries and immunoassays on the Roche cobas® 6000 analyzer—A CALIPER pilot study

OBJECTIVE The aim of this study was to determine age- and sex-specific pediatric reference intervals for 28 analytes on the Roche cobas 6000 analyzer. DESIGN AND METHODS The study was conducted at the Hospital for Sick Children in Toronto, Canada. Approximately 600 outpatient samples from a pediatric population deemed to be metabolically stable were subdivided into five age classes ranging from 0 to 20 years of age and further partitioned by gender. Reference intervals were established, after removal of samples significantly affected by hemolysis, icterus and lipemia and outlier exclusion, using the Robust statistical method to obtain the 2.5th and 97.5th percentiles. RESULTS Age (birth to 20 years of age) and gender-appropriate pediatric reference intervals for 28 analytes are reported. CONCLUSIONS These reference intervals provide the basis for clinical interpretation of laboratory results using the Roche cobas 6000 analyzer or related instrumentation/methods, provided adequate reference interval verification studies are performed.

Long-term stability of biochemical markers in pediatric serum specimens stored at − 80 °C: A CALIPER Substudy

OBJECTIVES Pediatric serum samples collected from healthy children in the CALIPER (Canadian Laboratory Initiative in Pediatric Reference Interval) project are stored at -80 °C for various periods of time. This study aimed to determine the stability of chemistry, protein, and hormone analytes under these conditions. DESIGN AND METHODS Serum samples collected from children of 0-18 years of age attending outpatient clinics were pooled into a single pool or into age-group specific pools. Following baseline measurement, each pool was aliquoted and kept frozen at -80 °C until analysis. Samples were analyzed for 57 biochemical markers at monthly intervals over a 10-13 month period and each aliquot was subject to one freeze-thaw cycle before analysis. The analysis was performed on VITROS Chemistry System, COBAS INTEGRA 400 Plus and IMMULITE 2500. Values obtained at monthly intervals were compared to baseline measurements and examined for trends over time. RESULTS A majority of analytes measured in this study showed no significant time-dependent change relative to baseline or trend over time after up to 13 months of storage. PTH showed up to 27.2% decline after 10 months of storage with most of the decline evident after 2 months. Most analytes showed variability over time, which is thought to reflect assay variability rather than changes in analyte stability. CONCLUSIONS The study shows stability for a majority of analytes stored in serum at -80 °C after up to 13 months of storage. Samples do not require immediate testing for reference interval determination for the selected analytes with possible exception of PTH.

Analytical evaluation of the VITROS ® 5600 Integrated System in a pediatric setting and determination of pediatric reference intervals

OBJECTIVES To evaluate the VITROS 5600 Integrated System in a pediatric setting and to determine age- and gender-specific pediatric reference intervals for several common analytes. DESIGN AND METHODS The instrument was evaluated using QC material and patient samples. Reference intervals were determined using samples obtained from children attending select outpatient clinics. RESULTS Imprecision analysis for 25 analytes and serum indices, the turnaround time for a simulated workload, and MicroSensor performance were assessed in our pediatric laboratory. Pediatric reference intervals for 25 analytes were also determined according to the CLSI/IFCC C28-A3 guidelines using 770 samples and over 15,000 analyses. CONCLUSION The VITROS 5600 Integrated System is suitable for use in a pediatric setting. Age- and gender-partitioned pediatric reference intervals for 25 common analytes were also determined as a pilot to the ongoing CALIPER project. These reference intervals are valuable for all VITROS users as well as any laboratory assessing these analytes once they demonstrate the acceptability of transference to their laboratory.

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.

Influence of fasting and sample collection time on 38 biochemical markers in healthy children: A CALIPER substudy

OBJECTIVES Fasting samples can be difficult to obtain in the pediatric setting, particularly in neonates. As part of the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER), we aimed to determine if there are differences in serum concentrations of pediatric biochemical markers measured at fasting, postprandial, and random time points throughout the day. DESIGN AND METHODS Blood was drawn from 27 healthy children and adolescents (aged 4-18) with informed consent at 4 time points: after overnight fast, mid-morning after breakfast, within 2h after lunch, and late afternoon. The effect of fasting on 38 chemistries was evaluated by paired, two-tailed studentst-tests. Analysis of the effect of time of day was done using paired, repeated-measures ANOVA. RESULTS Fasting significantly affected 22 analytes, with HDL cholesterol being the most highly affected. Values tended to decrease postprandially, except for five analytes, including triglycerides, which increased. By ANOVA, 28 chemistries significantly differed across times of day tested. CONCLUSIONS Fasting is necessary for analysis of certain chemistries in pediatric subjects. Pediatricians should consider diurnal factors when ordering non-fasting tests and interpreting test results.