Reference Intervals For Children Research Articles

Advanced lipid testing in children: a call for pediatric reference intervals

Abstract Introduction Abnormal serum lipid levels in childhood are associated with dyslipidemia and increased risk of cardiovascular disease in adulthood. High prevalence of childhood obesity in the US has increased concerns about the adverse effects of abnormal lipids in children, prompting recommendations for universal lipid screening in adolescents. While specialized analysis of lipoproteins by nuclear magnetic resonance (NMR) has been predominately used in adults with risk factors for CVD, our laboratory also receives pediatric samples for NMR testing, most commonly associated with diagnostic codes for hyperlipidemia and/or abnormal weight gain. Currently adult-based reference ranges for the NMR lipoprotein measurements are used in children. The objective of this pilot study was to compare lipoprotein profiles of healthy adults and children and to assess the impact of reference intervals for children on clinical interpretation of pediatric results. Methods Serum samples were collected from 60 healthy children, ages 1 – 17 years, and 148 healthy adults, ages 19 – 64 years old. NMR lipoprotein analysis was performed using the Axinon lipoFIT by NMR assay on a 600 MHz NMR (Bruker). Statistical analysis was conducted in Excel and GraphPad Prism. Reference intervals (RIs) were determined in EP Evaluator as the central 95% interval. Results The levels of atherogenic low-density lipoprotein particle number (LDL-P) and small LDL-P (sLDL-P) were significantly lower in children compared to adults: 783±222 vs 1212±412 nmol/L and 302±142 vs 498±306 nmol/L, respectively (p<0.00001). Additionally, LDL size (LDL-s) was significantly larger in children than adults (21.23±0.29 vs 21.06±0.44 nm, p=0.006), reflecting prevalence of less dense LDL-P in the former group. Pediatric samples also had lower number of anti-atherogenic high-density lipoprotein particles (HDL-P) compared to adults: 29.4±4.6 vs 33.5±4.6 µmol/L, p<0.00001. The significant differences in the number and size of lipoprotein particles between adults and children implies that currently used adult reference intervals might not be appropriate for evaluating pediatric NMR samples. Therefore, we estimated RIs for our heathy pediatric and adult cohorts and applied these RIs to our retrospective data of 618 samples from children 1-17 years old, which were submitted for NMR lipoprotein testing in recent years. The analysis showed that using the adult RIs, 37% and 27% of pediatric samples would not flag for abnormally elevated LDL-P and sLDL-P, respectively; but they would be flagged based on the RI derived from our pediatric cohort. Moreover, 12% pediatric samples flagged for low HDL-P would have normal HDL-P levels if pediatric RIs are used instead of adult RIs. Conclusions Use of adult reference intervals in children may result in misclassification of lipid status and underestimation or overestimation of dyslipidemia and CVD risk in children. This pilot study highlights the need for pediatric reference intervals to guide interpretation of advanced lipid testing in children.

A-227 One Step Toward Implementation of Advanced Lipoprotien Testing in Children: A Pilot Study on Pediatric Reference Ranges

Abstract Background Abnormal serum lipid levels in childhood are associated with dyslipidemia and increased risk of cardiovascular disease in adulthood. High prevalence of childhood obesity in the US has increased concerns about the adverse effects of abnormal lipids in children, prompting recommendations for universal lipid screening in adolescents. While specialized analysis of lipoproteins by nuclear magnetic resonance (NMR) has been predominately used in adults with risk factors for CVD, our laboratory also receives pediatric samples for NMR testing, most commonly associated with diagnostic codes for hyperlipidemia and/or abnormal weight gain. Currently adult-based reference ranges for the NMR lipoprotein measurements are used in children. The objective of this pilot study was to compare lipoprotein profiles of healthy adults and children and to assess the impact of reference intervals for children on clinical interpretation of pediatric results. Methods Serum samples were collected from 60 healthy children, ages 1 - 17 years, and 148 self-identified healthy adults, ages 19 - 64 years old. NMR lipoprotein analysis was performed using the Axinon lipoFIT by NMR assay on a 600 MHz NMR (Bruker). Statistical analysis was conducted in Excel and Graph Pad Prism. Reference intervals (RIs) were determined in EP Evaluator as the central 95% interval. Results The levels of atherogenic low-density lipoprotein particle number (LDL-P) and small LDL-P (sLDL-P) were significantly lower in children compared to adults: 783±222 vs 1212±412 nmol/L and 302±142 vs 498±306 nmol/L, respectively (p<0.00001). Additionally, LDL size (LDL-s) was significantly larger in children than adults (21.23±0.29 vs 21.06±0.44 nm, p=0.006), reflecting prevalence of less dense LDL-P in the former group. Pediatric samples also had lower number of anti-atherogenic high-density lipoprotein particles (HDL-P) compared to adults: 29.4±4.6 vs 33.5±4.6 µmol/L, p<0.00001. The significant differences in the number and size of lipoprotein particles between adults and children implies that currently used adult reference intervals might not be appropriate for evaluating pediatric NMR samples. Therefore, we estimated RIs for our heathy pediatric and adult cohorts and applied these RIs to our retrospective data of 618 samples from children 1-17 years old, which were submitted for NMR lipoprotein testing in recent years. The analysis showed that using the adult RIs, 37% and 27% of pediatric samples would not flag for abnormally elevated LDL-P and sLDL-P, respectively; but they would be flagged based on the RI derived from our pediatric cohort. Moreover, 12% pediatric samples flagged for low HDL-P would have normal HDL-P levels if pediatric RIs are used instead of adult RIs. Conclusions Use of adult reference intervals in children may result in misclassification of lipid status and underestimation or overestimation of dyslipidemia and CVD risk in children. This pilot study highlights the need for pediatric reference intervals to guide interpretation of advanced lipid testing in children.

Reference intervals and stability of haptocorrin and holotranscobalamin in Danish children and elderly

BackgroundHaptocorrin (HC) and holotranscobalamin (holoTC) carry vitamin B12 (B12) in the circulation and can be useful biomarkers for evaluating B12 status. The concentration of both proteins depends on age, but data on reference intervals for children and the elderly are sparse. Similarly, not much is known about the effect of preanalytical factors. MethodsHC plasma samples from healthy elderly > 65 years (n = 124) were analysed, and both HC and holoTC were analysed in paediatric serum samples ≤ 18 years (n = 400). Furthermore, we investigated assay precision and stability. ResultsHC and holoTC were effected by age. We established reference intervals for HC: 2–10 years, 369–1237 pmol/L; 11–18 years, 314–1128 pmol/L; 65–82 years, 242–680 pmol/L and for holoTC: 2–10 years, 46–206 pmol/L; 11-18 years, 30–178 pmol/L. Analytical coefficients of variations of 6.0–6.8% and 7.9–15.7% were found for HC and holoTC, respectively. HC were affected when stored at room temperature and by freeze/thaw. HoloTC was stable at room temperature and after delayed centrifugation. ConclusionWe present novel 95% age-related reference limits for HC and HoloTC in children, and for HC both in children and elderly. Moreover, we found HoloTC to be fairly stable when stored, whereas HC was more vulnerable to preanalytical factors.

Highly sensitive tandem mass spectrometric measurement of serum estradiol without derivatization and pediatric reference intervals in children and adolescents.

Monitoring estradiol (E2) is important for determining the onset of pubertal development as well as in the evaluation of girls with precocious puberty. However, E2 measurement remains an analytical challenge in children, who have lower circulating levels. We developed and evaluated a simple and sensitive LC-MS/MS procedure for serum E2 quantification in pediatric populations and established age- and sex-specific pediatric reference intervals. Residual patient serum samples were used to evaluate the analytical performance of our in-house LC-MS/MS E2 assay. The evaluation included accuracy, precision, linearity, functional sensitivity (LLoQ), and method comparison. Age- and sex-specific pediatric E2 reference intervals were also established from a cohort of 405 healthy children (birth to 18 years) recruited with informed consent. Age- and sex-specific differences were assessed, and outliers were removed. Reference intervals were established using the robust method. The assay imprecision was<5.3 %. Assay linearity ranged from 13.7 to 1923.3 pmol/L. The LLoQ corresponding to a CV of 20 % was determined to be 8.9 pmol/L. Bland-Altman analysis revealed a mean bias of 29.3 pmol/L or 9.1 % between our LC-MS/MS E2 assay and an external reference laboratory measuring E2 by LC-MS/MS. Our LC-MS/MS E2 assay shows acceptable accuracy, precision, functional sensitivity (LLoQ), and linearity for E2 quantification. Our LC-MS/MS E2 assay also showed good agreement with an external reference laboratory measuring E2 by LC-MS/MS. In addition, using CALIPER samples, we established robust age- and sex-specific pediatric E2 reference intervals to improve accuracy of test result interpretation and clinical decision making.

Data mining for prothrombin time and international normalized ratio reference intervals in children.

Reference intervals (RIs) help physicians in differentiating healthy from sick individuals. The prothrombin time (PT) and International normalized ratio (INR) fluctuate in coagulation pathway defects and have interlaboratory variability due to the instrument/reagent used. As direct method is difficult in children, we chose an indirect data mining method for the determining PT/INR RIs. The indirect method overcomes the substantial financial and logistic challenges, and ethical restrictions in children, moreover, allows partitioning in more fine-grained age groups. Prothrombin Time/INR measurements performed in patients aged birth-18 years between January 2013 and December 2020, were retrieved from laboratory management system of the Aga Khan Hospital. Reference intervals were computed using an indirect KOSMIC algorithm. The KOSMIC package function on the assumption that the non-pathologic samples follow a Gaussian distribution (after Box-Cox transformation of the data), following an elaborate statistical process to isolate distribution of physiological samples from mixed dataset. A total of 56,712 and 52,245 values were retrieved for PT and INR respectively. After the exclusion of patients with multiple specimens obtained during the study period, RIs were calculated for 37,356 (PT) and 37,192 (INR) children with stratification into 9 age groups. A comparison of 2.5th and 97.5th percentile results with those of established RIs from SickKids Handbook of Pediatric Thrombosis and Hemostasis demonstrated good agreement in between different age groups. This study supports data mining as an alternate approach for establishing PT/INR RIs, specifically in resource-limited settings. The results obtained are specific to studied population and instrument/reagent used. The study also allows understanding of fluctuations in coagulation pathways with increasing age and hence better clinical decision-making based on PT and INR results.

The Androstenedione Roche Elecsys immunoassay has superior comparability to the LC-MS/MS assay than the Siemens Immulite immunoassay

Androstenedione (ASD) is a biomarker used in the diagnostic workup of hyperandrogenism, congenital adrenal hyperplasia, premature adrenarche, and polycystic ovary syndrome (PCOS). The Elecsys ASD competitive electrochemiluminescence immunoassay (Roche Diagnostics, Indianapolis, IN) is a new assay recently available in the US. ObjectiveThis study evaluated the analytical and clinical performance of the Elecsys ASD assay. Design & MethodsWe evaluated the linearity/analytical measuring range (AMR), precision, and accuracy of the Elecsys ASD assay on the cobas e601 analyzer. ASD was measured in serum/plasma in the Elecsys ASD, Immulite (Siemens Medical Solutions USA, Inc. Malvern, PA), and LC-MS/MS assays. Reference intervals (RI) were evaluated across genders, menopausal status, and in children. Statistical analysis was performed using EP evaluator and R program. ResultsThe Elecsys ASD assay had a linear response across the AMR. The intra- and inter-assay coefficients of variation at various concentrations were ≤4.5%. The Elecsys ASD assay had a mean difference of −0.04 ng/mL (−1.7%) with the LC-MS/MS assay, whereas the Immulite assay had a mean difference of 1.17 ng/mL (66%) and −1.22 ng/mL (−38%) compared to the LC-MS/MS and Elecsys ASD assays, respectively. The Roche recommended RIs for healthy men (0.280–1.52 ng/mL) and postmenopausal women (0.187–1.07 ng/mL) were successfully verified. The RIs for children were adopted from published data. For pre-menopausal women, a RI of <1.60 ng/mL was established. The ASD concentrations in women with and without PCOS overlapped. ConclusionsThe Elecsys ASD assay has superior comparability to the LC-MS/MS assay than the Immulite assay.

Age-dependent reference intervals in children for the disialoganglioside, GD2, a circulating tumor biomarker for the childhood cancer, neuroblastoma

BackgroundThe disialoganglioside GD2 is a circulating tumor biomarker for the childhood cancer, neuroblastoma. This study establishes reference intervals for GD2 concentration in children within the age range where neuroblastoma commonly occurs. MethodsLeftover plasma samples taken for routine clinical laboratory tests from children without cancer were collected and assayed for the 18-carbon fatty acid chain length lipoform of GD2 using a validated high-pressure liquid chromatography tandem mass spectrometry method with a lower limit of quantification of 3 nM. Samples were stratified into 5 age cohorts (0–6 months, 6–12 months, 12–36 months, 3–10 years and > 10 years). Non-parametric statistical methods were used to define the upper bound of the reference interval for each age cohort. ResultsGD2 was measurable in 90% of samples from children < 10 years of age and GD2 concentration was age-dependent, peaking at 9 months followed by a gradual decline. GD2 was below the lower limit of quantification in 55% of samples in the > 10 years cohort. Upper bounds of reference intervals were 15.5 nM in 0–6 month cohort, 35.1 nM in 6–12 month cohort, 24.9 nM in 12–36 month cohort, 18.4 in 3–10 year cohort and 10.4 nM in > 10 year cohort. ConclusionsAge-dependent reference intervals were defined for circulating GD2 in children. GD2 concentration was highest in the 6–12 month age cohort, which is below the age of most children with high-risk neuroblastoma. The peak GD2 concentration at 9 months may reflect neurodevelopmental events in the brain.

Reference interval for type III procollagen (PIIINP) using the Advia centaur PIIINP assay in adults and elderly

Objective The amino-terminal peptide of type III procollagen (PIIINP) is a byproduct of type III collagen synthesis that exhibits promise as a biomarker of fibrosis, specifically in monitoring hepatic fibrosis in methotrexate treated patients. The Advia Centaur® PIIINP assay is developed for track-based automated laboratory systems and is suitable for large volume analysis. Reference intervals in children and younger adults have been published previously. Here we measured PIIINP to determine reference ranges, specifically including elderly patients, for whom such are currently lacking. Methods Samples were collected from subjects ranging from 20 to 98 years of age. Blood donors and clinical samples from primary care patients were used for reference interval calculation. Samples were analysed using the Advia Centaur® PIIINP assay. After exclusion of samples high in alanine transaminase (AST), aspartate transaminase (ALT), and C-reactive protein (CRP) 386 samples were used in the reference interval calculation. Results and conclusion We determined the following reference interval for the Advia Centaur® PIIINP assay: the lower limit of the reference interval (2.5% percentile with 95% CI) was 4.42 (4.20–4.65) µg/L and the upper limit of the reference interval (97.5% percentile 95% CI) 16.0 (15.04–17.02) µg/L.No significant differences in mean PIIINP concentrations were found between men and women. While differing mean PIIINP concentrations were seen among subjects in different age groups, the differences were small and partitioning of reference range was determined not to be necessary.

Data mining of pediatric reference intervals

Abstract Laboratory tests are essential to assess the health status and to guide patient care in individuals of all ages. The interpretation of quantitative test results requires availability of appropriate reference intervals, and reference intervals in children have to account for the extensive physiological dynamics with age in many biomarkers. Creation of reference intervals using conventional approaches requires the sampling of healthy individuals, which is opposed by ethical and practical considerations in children, due to the need for a large number of blood samples from healthy children of all ages, including neonates and young infants. This limits the availability and quality of pediatric reference intervals, and ultimately negatively impacts pediatric clinical decision-making. Data mining approaches use laboratory test results and clinical information from hospital information systems to create reference intervals. The extensive number of available test results from laboratory information systems and advanced statistical methods enable the creation of pediatric reference intervals with an unprecedented age-related accuracy for children of all ages. Ongoing developments regarding the availability and standardization of electronic medical records and of indirect statistical methods will further improve the benefit of data mining for pediatric reference intervals.

Thyroid panel reference intervals in healthy children and adolescents: A Spanish cohort

ObjectivesIn children, thyroid hormones are essential for correct physical and neurological development. The recommended process for defining reference intervals (RIs) is the direct approach; however, indirect methods are an effective alternative. This study aimed to explore age- and sex-dependent relationships between serum concentrations of thyroid hormones in a large population-based cohort of healthy Spanish Caucasian children and calculate RIs. Material and MethodsResults of serum thyroid-stimulating hormone (TSH) and free thyroxine (fT4) were collected from laboratory data of N (TSH = 23201; fT4 = 20728) patients aged 1 month − 15 years. These results were validated with a prospective study.Analyses of serum concentrations of TSH and fT4 were performed on ARCHITECT i2000 (Abbott Diagnostics, US). Percentiles (2.5th to 97.5th) were determined for each variable and taken as the RI. ResultsNo difference was found between serum TSH concentrations in male and female children of all age groups. A difference between serum fT4 concentrations in males and females and an age-dependent correlation for both sexes were found. ConclusionThere is very little consensus on RIs in children. Our data confirm it is possible to use data mining techniques to calculate reliable and clinically useful RIs.

Establishing reference intervals of 20 biochemical markers for children in Southwestern Fujian, China based on the UniCel DxC 800 system

To date, China has no industry standard for reference intervals of paediatric blood biochemical markers. This study aimed to evaluate changes in biochemical markers in the venous blood of healthy children aged 29 days to 12 years, derived from the UniCel DxC 800 system, and establish appropriate reference intervals. We analysed venous blood from 1980 healthy children for 20 biochemical markers. Reference intervals were established according to the Clinical and Laboratory Standards Institute C28-A3c guideline and compared with those of adults in China. All markers except for sodium and chlorine required partitioning by age, but not by sex. The reference intervals of total protein, albumin, globulin, carbon dioxide, urea nitrogen, creatinine and uric acid consistently increased with age in children, but were always lower than those of adults. Children aged 29 days to 12 years had a single combined RI for sodium and chloride, respectively; although the reference intervals in children were similar to those of adults, their upper limits were lower. The reference intervals of direct bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, potassium, calcium, magnesium, and phosphorus continued to decline with age. The reference intervals of total bilirubin, indirect bilirubin, and gamma-glutamyl transpeptidase initially declined followed by a slight rebound. While establishing reference intervals, most markers required partitioning by age (aged 29 days to 12 years); the partitioning scheme differed for each marker, and paediatric reference intervals differed from those for adults. It is therefore necessary to establish separate paediatric reference intervals.

Continuous, complete and comparable NT-proBNP reference ranges in healthy children.

NT-proBNP is one of the most important biomarkers for the diagnosis and risk assessment of heart failure in adults. Age- and gender-independent reference intervals (RIs) have been reported. In contrast, RIs in children are strongly age-dependent, do not exist for all ages and reveal a right-skewed distribution. Accordingly, no common Z-score can be formed and a cross-age interpretive method, so far, is missing. Within the paper on hand, new evaluation techniques are applied to already published NT-proBNP study results and additionally to newly gained data. Upper limits (ULs), lower limits (LLs) and 50th percentiles are tested for power-like behavior as a function of age using linear regression analysis. Functions for continuous RIs are derived and reference limits are calculated on a per day basis. A corresponding Zlog formula is deduced and its usefulness is stated in two clinical examples. The power-like behavior of NT-proBNP concentration from birth to 18 years is demonstrated. With age in days t and measured NT-proBNP value x in pg/mL, an age-specific Zlog value may directly be calculated using the equation:ZlogNT-proBNP=log x+0.512⋅log t-3.4171.489+0.014⋅log t⋅3.92${\rm{Zlo}}{{\rm{g}}_{{\rm{NT - proBNP}}}} = {{\log \;x + 0.512 \cdot \log \;t - 3.417} \over {1.489 + 0.014 \cdot \log \;t}} \cdot 3.92$. Using formulas for UL and LL, continuous RIs from 0 to 18 years may be obtained. Continuity corresponds to physiological changes in the body much better than discrete RIs. With the advent of an NT-proBNP-specific Zlog value, a cross-age Z-score equivalent is providing an easy interpretation aid in everyday pediatric practice. This new approach allows to identify clinical worsening much better, sooner and more clearly than previous absolute values.

Application of adult reference intervals in children

The aim of this study was to evaluate to which extend adult reference intervals (RIs) could be applied in children. A local paediatric population (aged 1 to < 20years), based on first draw samples from general practitioners (GPs), was established. Children with samples taken at a hospital or > 3 samples from GPs were excluded. Analytes evaluated included haematological, liver and pancreatic function, kidney function, electrolytes, and metabolism parameters. Applicability of adult RIs in children aged 1-17years was evaluated using individuals aged 18-19years as reference groups for the adult RIs. The local population consisted of 31,024 children with 282,721 analyses in total. For each analyte, 17 age strata and two gender strata were established. Partitioning was not warranted in 51% of the male strata and in 69% of the female strata. Adult RIs could be applied in 42% for children aged 1-< 10years, 57% for children aged 10-< 15years, and 85% for children aged 15-<18years.Conclusion: for certain analytes, there is no need to partition between adult and paediatric RIs, but a need for age- and gender-specific RIs remains for several clinical laboratory tests.What is Known:• Establishing paediatric reference intervals (RIs) is time consuming, costly, and not feasible for many laboratories. Transference of RIs established elsewhere often leads to misclassification of paediatric laboratory results.• Adult RIs are often more easily established and validated.What is New:• Adult RIs can be applied to children as young as 2years for some analytes. Conversely, for some analytes, adult RIs cannot be applied in children aged 1-17years.• Laboratory data can be applied in evaluating the need for partitioning in reference intervals.

Investigation and analysis of the reference intervals for complete blood cell counting in 110 maternal and child health service centers and children′s hospitals in China

Objective To investigate and analyze the source and upper and lower limits of the reference interval of children′s complete blood count in 110 maternal and child health hospitals and children′s hospitals nationwide. Methods Laboratories submitted the data of reference intervals via external quality assessment (EQA) software which was based on the web. To collect the results of reference intervals questionnaires on complete blood cell counting of children in laboratory departments of 110 maternal and child care service centers and children′s hospitals in China in 2017. Questionnaires include information on the source of reference intervals for 18 items of complete blood count, whether to verify, upper and lower limits, grouping, methods used, instruments, reagents, and calibrators. Data was analyzed using Microsoft Excel 2007 and SPSS 22.0. The median, P25, P75were obtained, and rank sum test were used to determine whether there were statistical differences between groups. Results The results of 110 laboratories were obtained after rejecting invalid data. The reference intervals were mainly derived from operating procedures and the laboratories themselves, of which 50.5%-53.6% of the laboratories were validated. The white blood cell counting reference intervals gradually decreased from birth to adolescence, and the value was close to that of adults. The reference intervals of red blood cell counting and hemoglobin were close to that of adults except in the neonatal period. The value of the reference intervals of hematocrit slightly decreased with age. The difference between RBC, Hgb, HCT in reference intervals between groups was statistically significant when grouped by gender (P<0.05) . The reference intervals of white blood cell counting were less grouped by gender (5 laboratories) , and the difference among groups was not statistically significant. There were no statistically significant differences in the reference intervals of elements between the two measurement systems that Sysmex XN series and Sysmex XS-800i/XS-1000i/XS 500i/XS 900i series. Conclusions The establishment of reference intervals for children′s complete blood cell counting was urgently needed. The reference intervals of the complete blood cell counting item had statistical significance in both age and gender. It was suggested that the industry standard of children′s complete blood cell counting reference intervals should be established based on age and gender. Key words: Blood cell count; Reference values; Child

Erythrocyte Oxidative Stress Markers in Children: A Clinical Laboratory Experience

Because oxidative damage to erythrocytes plays an important pathophysiologic role in many diseases and there are some difficulties in obtaining reference intervals for children in many laboratory tests, this work intends to describe the determination of oxidative stress parameters and the results obtained for children. A total of 280 blood samples were obtained from children between 8 and 11 years old, without any hematological disease. Samples were analyzed for seven oxidative stress parameters, methemoglobin, reduced glutathione, TBARS, percentage of hemolysis and activity of the enzymes G6PD, superoxide dismutase, and catalase. To draw a comparison, the same parameters were analyzed in children with sickle cell disease. Because all results presented normal distribution in the Kolmogorov-Smirnov, data were expressed as 2.5 and 97.5th accumulated percentiles and the statistical analysis between male and female children was performed using Student t -test. A p value

LC-MS/MS based assay and reference intervals in children and adolescents for oxysterols elevated in Niemann–Pick diseases

BackgroundNiemann–Pick type C (NP-C) is a rare progressive neurodegenerative lipid storage disorder with heterogeneous clinical presentation and challenging diagnostic procedures. Recently oxysterols have been reported to be specific biomarkers for NP-C but knowledge on the intra-individual variation and on reference intervals in children and adolescents are lacking. MethodsWe established a LC-MS/MS assay to measure Cholestane-3β, 5α, 6β-triol (C-triol) and 7-Ketocholesterol (7-KC) following Steglich esterification. To assess reference intervals and intra-individual variation we determined oxysterols in 148 children and adolescents from 0 to 18 years and repeat measurements in 19 of them. ResultsThe reported method is linear (r>0.99), sensitive (detection limit of 0.03 ng/mL [0.07 nM] for C-triol, and 0.54 ng/mL [1.35 nM] for 7-KC) and precise, with an intra-day imprecision of 4.8% and 4.1%, and an inter-day imprecision of 7.0% and 11.0% for C-triol (28 ng/ml, 67 nM) and 7-KC (32 ng/ml, 80 nM), respectively. Recoveries for 7-KC and C-triol range between 93% and 107%.The upper reference limit obtained for C-triol is 40.4 ng/mL (95% CI: 26.4–61.7 ng/mL, 96.0 nM, 95% CI: 62.8–146.7 nM) and 75.0 ng/mL for 7-KC (95% CI: 55.5–102.5 ng/mL, 187.2 nM, 95% CI: 138.53–255.8 nM), with no age or gender dependency. Both oxysterols have a broad intra-individual variation of 46%±23% for C-triol and 52%±29% for 7-KC. Nevertheless, all Niemann–Pick patients showed increased C-triol levels including Niemann–Pick type A and B patients. ConclusionsThe LC-MS/MS assay is a robust assay to quantify C-triol and 7-KC in plasma with well documented reference intervals in children and adolescents to screen for NP-C in the pediatric population. In addition our results suggest that especially the C-triol is a biomarker for all three Niemann–Pick diseases.

Establishment of age/gender-related serum cycstatin C reference intervals in children by transmission turbidimetry

Objective To establish pediatric serum CysC reference intervals based on the children's hospital laboratory data in Zhejiang Province and analyse the effects of CysC levels on age and gender. Methods CysC was one of tests of a routine biochemical screening panel employed for most outpatients and inpatients in the children's hospital, and 8 127 subjects (4 264 boys and 3 863 girls) were selected from 13 567 subjects from laboratory information system according to the exclusion criterion with seriously systemic diseases, cardiovascular diseases, kidney diseases, elevated values of creatinine, urea or ALT for which 1.5-fold upper limit, serum samples with hemolysis, icterus or lipid turbidity, same patients with non-first-time CysC results and living addresses which were not in Zhejiang. The serum CysC was determined by transmission turbidimetry on Roche DPP modular automatic biochemical analyzer. SPSS 17.0 software and EXCEL 2003 were employed for statistical analysis in this study. Results The serum CysC concentration was a Gaussian distribution after log transformation. The mean Log-transformated CysC lg (CysC) concentration of boys in five age groups ( ＜ 1 month, 1 to 3 months, 4 to 11 months, 1 to 2 years and 2 to 16 years) were 0. 224,0. 170,0. 112,0. 061, -0. 011 (mg/L,lg) respectively, and the mean Log-transformated CysC lg(CysC) concentration of girls in five age groups were 0. 222, 0. 164, 0. 089, 0. 057,-0. 010 ( mmg/L, lg) respectively, and no statistically significant differences between Ig( CysC ) and gender in five age groups were found ( t values were 0. 174, 0. 362, 0. 445, - 1. 464 and - 0. 093, respectively,and corresponding P values all were greater than 0. 05 ). The mean lg ( CysC ) concentrations in five age groups were 0. 222,0. 166,0. 100,0. 059, - 0. 010 ( mmg/L, lg), and significant differences between Lg ( CysC ) and ages by Analysis of Variance were observed ( F = 309. 785 and P = 0 in between-groups totally,P = 0 in any two groups). Serum CysC levels were highest in the age of ＜ 1 month, then declined to the age of 2 years and kept stable in the age of 2 to 16 years. The serum CysC reference intervals for children were as follows:0. 95 -2. 92 mg/L in the age of ＜ 1 month, 0. 81 -2.67 mg/L in the age of 1 to 3 months, 0. 65 -2.45 mg/L in the age of 4 tol2 months, 0. 56 -2. 35 mg/L in the age of 1 year and 0. 45 -2. 13 mg/L in the age of 2 to 16 years. Conclusions There is no significant effect on pediatric CysC levels with gender but close correlated with age below 2 years old. It is necessary to establish appropriate age-related reference intervals of serum CysC for efficiently evaluating renal function in local children. Key words: Cystatin C ; Reference interval; Data mining; Transmission turbidimetry; Pediatrics

Citrulline levels in a paediatric age group: Does measurement on dried blood spots have additional value?

Background Citrulline is considered to be a marker of absorptive enterocyte mass. Citrulline levels can be measured in plasma or dried blood spot (DBS) samples. The purpose of this study is to calculate reference intervals for plasma and DBS citrulline concentrations in children and to examine the effect of age and gender. Methods In 151 healthy subjects ranging from 1 month to 20 years of age, plasma and DBS citrulline concentration were determined by using Liquid Chromatography-tandem Mass Spectrometry. Citrulline concentrations were examined in relation to age and gender. Reference values were calculated according to the guidelines of the International Federation of Clinical Chemistry and the National Committee on Clinical Laboratory Standards. Results No significant influence of age and gender could be discerned on plasma or DBS citrulline concentration. In children, the reference intervals for citrulline bounded by the 2.5 and 97.5 percentiles are 13.31–69.05 μmol/L and 23.70–49.04 μmol/L for plasma and DBS samples respectively. Conclusions The reference intervals for citrulline levels in healthy children are widely dispersed. Measuring citrulline concentrations in dried blood spots delivers no additional value to plasma measurements for the calculation of reference intervals in children.

Pediatric reference intervals determined in ambulatory and hospitalized children and juveniles

Objectives The aim of this study was to determine reference intervals in children and juveniles with nine recently developed and widely used laboratory methods. Methods More than 800 ambulatory and hospitalized individuals of the University pediatry were carefully selected according to clinical status and chemical profile in an a posteriori process over a period of two and a half years. The reference group with well-balanced gender proportions and steady age distribution between 1 day and 17 years was subdivided in five age classes. The laboratory methods were: the enzyme methods ALT, AST, LDH and GGT, all reliably reference standardized with traceability to the IFCC reference methods at 37 °C; ALT and AST without pyridoxal-phosphate activation; ALP as not yet approved IFCC method; the soluble transferrin receptor (sTfR) and ferritin, the latter being the only heterogeneous procedure. Results The results confirm in most cases the typical age concentration relationship of the measured quantities documented for similar methods. In some critical cases, in particular for sTfR and ferritin, the study produces limits which differ distinctly from those earlier reported. Gender differentiation was outlined according to statistical calculations. Conclusion Proposals for reference intervals were derived from the central 95 percentile values.

Automated HPLC Assay for Urinary Collagen Cross-links: Effect of Age, Menopause, and Metabolic Bone Diseases

The pyridinium cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) are established markers of bone resorption. We evaluated the analytical and clinical performance of a commercially available PYD HPLC assay and established reference intervals in children and adults. We used a commercially available reagent set (Chromsystems Instruments & Chemicals) to measure PYD and DPD in 319 healthy controls (156 premenopausal women, 80 healthy men, and 83 healthy children age 1 month to 14 years) and 397 patients with metabolic bone diseases (postmenopausal osteoporosis, n = 175; male osteoporosis, n = 176; hyperparathyroidism, n = 17; hyperthyroidism, n = 19; Paget disease, n = 10). The mean intraassay and interassay CVs were <6% and <8% for both PYD and DPD, respectively. The reference interval was constant for premenopausal women in the age group 20-49 years. In men, cross-link values peaked at 20-29 years and decreased thereafter. Women with postmenopausal osteoporosis had significantly higher PYD (51%) and DPD (58%) values compared to premenopausal women. Similar results were found in osteoporotic men. In children the highest values were found in the first weeks and months after birth, followed by a decrease of 50%-60% at age 11-14 years. In metabolic bone diseases cross-link concentrations were significantly increased. The DPD:PYD ratio (mean value approximately 0.2) was remarkably constant in all populations evaluated. The automated HPLC assay is a precise and convenient method for PYD and DPD measurement. We established reference intervals for adult women and men and for children up to 14 years old. The cross-link concentrations we determined by use of this HPLC method confirm its clinical value in enabling identification of increased bone resorption in patients with metabolic bone diseases.