Age-dependent Reference Intervals Research Articles

Establishment of age- and -gender specific reference intervals for amino acids and acylcarnitines by tandem mass spectrometry in Turkish paediatric population.

We determined age- and gender-specific reference intervals (RIs) for acylcarnitines and amino acids by tandem mass spectrometry (MS/MS) in the Turkish paediatric population by using laboratory information system (LIS) data. A total of 9156 MS/MS results of children between 0-18 years of age, were downloaded from the LIS. Premature infants and newborns followed in the intensive care unit were excluded and only the first result of each patient attending outpatient clinics was included. Children with a known or suspected diagnosis of metabolic disease, malignancy, epilepsy, mental retardation, or genetic disorder were excluded. Laboratory results were evaluated and children with any pathological laboratory finding were excluded, resulting in a final sample size of 3357 (2029 boys and 1328 girls). Blood was collected by capillary puncture and spotted on Whatman 903 filter paper cards and analysed by MS/MS (Shimadzu LCMS-8050, Shimadzu Corporation, Kyoto, Japan). Data were evaluated for age and gender differences and age partitioning was performed according to the literature and visual evaluation of the data. Age subgroups were: ≤ 1 month, 2 months-1 year, 2-5 years, 6-10 years, and 11-18 years. There were significant age-related differences for the majority of amino acids and acylcarnitines thus age dependent RIs were established. Gender-specific RIs were established for tyrosine, leucine-isoleucine, isovalerylcarnitine (C5) and hexadecanoylcarnitine (C16). Establishing age-related RIs can enhance the quality of medical care by facilitating early diagnosis and therapy, especially in certain metabolic disorders presenting with mild biochemical abnormalities and subtle clinical manifestations.

A sensitive liquid chromatography–tandem mass spectrometry method for determination biomarkers of monoamine neurotransmitter disorders in cerebrospinal fluid

BackgroundCerebrospinal fluid (CSF) monoamine neurotransmitters, their precursors and metabolites are essential biomarkers in the diagnosis and follow-up of monoamine neurotransmitter disorders (MNDs). However, their extra low concentrations and potential instability challenge the detection method. Here, we present a method that enables simultaneous quantification of these biomarkers. MethodWith propyl chloroformate /n-propanol, 16 biomarkers in 50 μL of CSF were derivatized in situ within seconds under an ambient temperature. The derivatives were extracted by ethyl acetate and separated by a reverse phase column followed by mass spectrometric detection. The method was fully validated. Optimal conditions for standard solution preparation and storage, as well as CSF sample handling, were investigated. CSF samples from 200 controls and 16 patients were analyzed. ResultsThe derivatization reaction stabilized biomarkers and increased sensitivity. Most biomarkers were quantifiable in concentrations between 0.02 and 0.50 nmol/L that were sufficient to measure their endogenous concentrations. The intra- and inter-day imprecision were < 15% for most analytes, and accuracy ranged from 90.3% to 111.6%. The stability study showed that standard stock solutions were stable at −80 °C for six years when prepared in the protection solutions; Analytes in CSF samples were stable for 24 h on wet ice and at least two years at −80 °C; But repeated freeze-thaw should be avoided. With this method, age-dependent reference intervals for each biomarker in the pediatric population were established. Patients with MNDs were successfully identified. ConclusionThe developed method is valuable for MNDs diagnosis and research, benefiting from its advantages of sensitivity, comprehensiveness, and high throughput.

A visualization tool for continuous reference intervals based on GAMLSS

Abstract Reference intervals are an important component in the interpretation of medical laboratory findings. Especially in children and adolescents, their limits sometimes can change very rapidly with age. We suggest continuous methods to better represent the age-dependent progression of reference intervals. The generalized additive models for location, scale, and shape parameters (GAMLSS) from the R package gamlss generates continuous percentile plots of laboratory values. A user-friendly Shiny application called AdRI_GAMLSS (Age-dependent Reference Intervals), available at github.com/SandraKla/AdRI_GAMLSS, has been developed for this purpose. Using alkaline phosphatase (ALP) as an example, we obtain different smoothed percentile curves depending on the model used. We demonstrate the superiority of continuously modeled reference intervals compared to fixed age groups and provide the Shiny application AdRI_GAMLSS to make the technique easily accessible to clinicians and other experts.

Improved diagnostics of purine and pyrimidine metabolism disorders using LC-MS/MS and its clinical application.

To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify 41 different purine and pyrimidine (PuPy) metabolites in human urine to allow detection of most known disorders in this metabolic pathway and to determine reference intervals. Urine samples were diluted with an aqueous buffer to minimize ion suppression. For detection and quantification, liquid chromatography was combined with electrospray ionization, tandem mass spectrometry and multiple reaction monitoring. Transitions and instrument settings were established to quantify 41 analytes and nine stable-isotope-labeled internal standards (IS). The established method is precise (intra-day CV: 1.4-6.3%; inter-day CV: 1.3-15.2%), accurate (95.2% external quality control results within ±2 SD and 99.0% within ±3 SD; analyte recoveries: 61-121%), sensitive and has a broad dynamic range to quantify normal and pathological metabolite concentrations within one run. All analytes except aminoimidazole ribonucleoside (AIr) are stable before, during and after sample preparation. Moreover, analytes are not affected by five cycles of freeze-thawing (variation:-5.6 to 7.4%), are stable in thymol (variation:-8.4 to 12.9%) and the lithogenic metabolites also in HCl conserved urine. Age-dependent reference intervals from 3,368 urine samples were determined and used to diagnose 11 new patients within 7 years (total performed tests: 4,206). The presented method and reference intervals enable the quantification of 41 metabolites and the potential diagnosis of up to 25 disorders of PuPy metabolism.

Mixture density networks for the indirect estimation of reference intervals

BackgroundReference intervals represent the expected range of physiological test results in a healthy population and are essential to support medical decision making. Particularly in the context of pediatric reference intervals, where recruitment regulations make prospective studies challenging to conduct, indirect estimation strategies are becoming increasingly important. Established indirect methods enable robust identification of the distribution of “healthy” samples from laboratory databases, which include unlabeled pathologic cases, but are currently severely limited when adjusting for essential patient characteristics such as age. Here, we propose the use of mixture density networks (MDN) to overcome this problem and model all parameters of the mixture distribution in a single step.ResultsEstimated reference intervals from varying settings with simulated data demonstrate the ability to accurately estimate latent distributions from unlabeled data using different implementations of MDNs. Comparing the performance with alternative estimation approaches further highlights the importance of modeling the mixture component weights as a function of the input in order to avoid biased estimates for all other parameters and the resulting reference intervals. We also provide a strategy to generate partially customized starting weights to improve proper identification of the latent components. Finally, the application on real-world hemoglobin samples provides results in line with current gold standard approaches, but also suggests further investigations with respect to adequate regularization strategies in order to prevent overfitting the data.ConclusionsMixture density networks provide a promising approach capable of extracting the distribution of healthy samples from unlabeled laboratory databases while simultaneously and explicitly estimating all parameters and component weights as non-linear functions of the covariate(s), thereby allowing the estimation of age-dependent reference intervals in a single step. Further studies on model regularization and asymmetric component distributions are warranted to consolidate our findings and expand the scope of applications.

Establishment of reference intervals of homocysteine, cysteine, and methionine in apparently healthy Chinese adults

Background It has been widely reported that the key biomarkers of homocysteine metabolic pathways have emerged as markers or independent risk factors for cardiovascular disease and many other diseases. To improve the accuracy of evaluation in China, we establish the reference intervals of serum homocysteine (Hcy), cysteine (Cys), and methionine (Met) in apparently healthy Chinese adults. Methods We examined 1553 subjects aged ≥20 years who underwent Hcy metabolic pathway tests, in the Chaoyang Central Hospital between January 2019 and December 2020. The serum Hcy, Cys, and Met levels were simultaneously determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Multiple regression analysis was performed to identify sources of variation. A nonparametric method was used to establish the reference intervals. Results Gender differences were observed in Hcy and Met, with higher levels in males. Reference intervals for males were 6.09–17.00 μmol/L (Hcy) and 19.31–33.68 μmol/L (Met); conversely, the reference intervals for females were 4.61–14.61 μmol/L (Hcy) and 16.16–30.35 μmol/L (Met). The serum Cys reference intervals were seen to be gender-independent, but age-dependent reference intervals were needed; those for Cys were 188.85–334.27 μmol/L, 200.48–340.66 μmol/L, and 216.03–349.67 μmol/L in <40, 40–50, and ≥50 years, respectively. Conclusions By this study, the gender- or age-specific reference intervals of Hcy, Cys, and Met that were matched with the Chinese population were established, providing valuable references for clinical work and laboratory researches.

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.

Continuous reference intervals for pediatric testosterone, sex hormone binding globulin and free testosterone using quantile regression

Testosterone (T), sex hormone binding globulin (SHBG), free testosterone (FT) and bioavailable testosterone (BAT) are commonly employed tests in pediatric endocrinology and all require age-dependent reference intervals for interpretation. The common methods used to derive these reference intervals require decisions about data shape and/or age partition thresholds, which can result in sharp differences between age groups, particularly for pubescent children. Partitioning also results in a form of data loss, where data from one age-bin is completed disconnected from the adjacent age-bins. Non-parametric continuous reference intervals methods have previously been developed to avoid some of these drawbacks. These strategies use all the available data and smooth transitions between ages avoiding partitioning. However, the fitting process involves selection and adjustment of many parameters and it can be difficult to maintain a reproducible approach. Here we provide a workflow for non-parametric continuous reference intervals applied to T, FT, BAT, and SHBG using the R language quantregGrowth package. T measurements were determined by LC-MS/MS, FT and BAT were calculated, and SHBG was measured on the Roche Cobas e601. The continuous interval methodology is described in detail with code examples and illustrations for reproducibility.

Use of data mining in the establishment of age-adjusted reference intervals for parathyroid hormone

Background-AimAppropriately defined reference values are of vital importance for the correct interpretation of laboratory results. However, the implementation of in-house reference intervals by traditional methods is rare due to difficulties in the selection of reference individuals, so indirect methods are often alternatively used.Parathyroid hormone (PTH) is a crucial hormone for the metabolism of calcium and phosphorus whose result can lead to erroneous diagnoses and medical actions if adequate reference intervals are not readily available.Our goal was to obtain reference values ​​for adult population for PTH by using data mining. Based on possible new reference intervals, we also aimed to perform a retrospective evaluation of the individuals mistakenly classified. MethodsRetrospective observational study between 2014 and 2019. All requests from individuals aged ≥ 18 years were considered if PTH was requested together with serum creatinine, 25-hydroxyvitamin D, calcium, albumin and phosphate (within their respective reference intervals). PTH was measured on the Cobas e411 platform (Roche).Requests were grouped by age and sex. Differences among partitions were assessed by the Harris-Boyd’s test and reference intervals were determined by the non-parametrical calculation of percentiles 2.5 and 97.5. ResultsA total of 2279 laboratory requests were included. Reference intervals for PTH were seen to be sex-independent, but age-dependent reference intervals were needed: 18–39, 40–59 and >60 years.Based on the reference intervals obtained, up to 20.1% of individuals were misclassified. ConclusionsAppropriateness of PTH reference intervals would lead to a reduction in the number of additional tests and avoid wrong medical actions, thus improving not only patient safety, but also the healthcare system as a whole.

Impact of age, body weight and metabolic risk factors on steroid reference intervals in men.

To evaluate the independent impact of age, obesity and metabolic risk factors on 13 circulating steroid levels; to generate reference intervals for adult men. Cross-sectional study. Three hundred and fifteen adults, drug-free and apparently healthy men underwent clinical and biochemical evaluation. Thirteen steroids were measured by LC-MS/MS and compared among men with increasing BMI. Moreover, the independent impact of age, BMI and metabolic parameters on steroid levels was estimated. Upper and lower reference limits were generated in steroid-specific reference sub-cohorts and compared with dysmetabolic sub-cohorts. We observed lower steroid precursors and testosterone and increase in estrone levels in men with higher BMI ranges. By multivariate analysis, 17-hydroxyprogesterone and dihydrotestosterone decreased with BMI, while cortisol decreased with waist circumference. Estrone increased with BMI and systolic blood pressure. Testosterone decreased with worsening insulin resistance. 17-hydroxypregnenolone and corticosterone decreased with increasing total/HDL-cholesterol ratio. Age-related reference intervals were estimated for 17-hydroxypregnenolone, DHEA, 17-hydroxyprogesterone, corticosterone, 11-deoxycortisol, cortisol and androstenedione, while age-independent reference intervals were estimated for progesterone, 11-deoxycorticosterone, testosterone, dihydrotestosterone, estrone and estradiol. Testosterone lower limit was 2.29 nmol/L lower (P = 0.007) in insulin resistant vs insulin sensitive men. Furthermore, the upper limits for dihydrotestosterone (-0.34 nmol/L, P = 0.045), cortisol (-87 nmol/L, P = 0.045-0.002) and corticosterone (-10.1 nmol/L, P = 0.048-0.016) were lower in overweight/obese, in abdominal obese and in dyslipidaemic subjects compared to reference sub-cohorts, respectively. Obesity and mild unmedicated metabolic risk factors alter the circulating steroid profile and bias the estimation of reference limits for testosterone, dihydrotestosterone, cortisol and corticosterone. Applying age-dependent reference intervals is mandatory for steroid precursors and corticosteroids.

In Search of Normality for Vitamin K1: Establishing Age-Dependent Reference Intervals in the Danish Population.

A growing body of evidence suggests that vitamin K has beneficial effects on human health, especially cardiovascular and bone health. Vitamin K1 (phylloquinone), the predominant form of vitamin K in blood, is regarded as an indicator of vitamin K status, but to our knowledge no reference intervals (RIs) have been established for vitamin K1. In this population-based study, vitamin K1 was measured in serum from 3808 Caucasian individuals without diabetes from 26 to 78 years of age. The need for gender- and age-partitioned vitamin K1 reference intervals was evaluated using Lahti's method, and exclusion criteria were defined to obtain as healthy a study group as possible. The excluded subgroups were tested for differences in mean serum vitamin K1 levels. Serum vitamin K1 levels were quantified using an in-house newly developed, validated, and highly sensitive online SPE-LC-MS/MS method with a limit of quantitation of (LOQ) 0.05 nmol/L. The reference interval for serum vitamin K1 was 0.22 to 3.95 nmol/L for individuals aged 26 to 44 years and 0.35 to 3.70 nmol/L for individuals aged 45 to 78. Similar age-specific reference intervals were established for vitamin K1-triglyceride ratio being 0.20 to 3.16 and 0.31 to 3.44, respectively. No significant difference was found between genders. Serum vitamin K1 was detectable in all serum samples. Individuals with known comorbidity were found to have significantly lower serum vitamin K1 compared to those without comorbidity. Current smokers had lower serum vitamin K1 compared to nonsmokers. Age-dependent reference intervals were established for serum vitamin K1 and vitamin K1-triglyceride ratio in a well-defined, healthy Caucasian population. Lower serum vitamin K1 levels were found in individuals with known comorbidity, suggesting an association between serum vitamin K1 and disease status. Further studies are needed to determine an optimal serum vitamin K1 level.

N-terminal pro-B-type natriuretic peptide in amniotic fluid of fetuses with known or suspected cardiac load

BackgroundMyocardial dysfunction occurs in a variety of fetal disorders. Findings from adult cardiology, where n-terminal pro-B-type natriuretic peptide (nt-proBNP) is an established biomarker of left ventricular dysfunction have been extended to fetal life. Since fetal blood sampling is technically challenging we investigated amniotic fluid nt-proBNP for its suitability to diagnose fetal myocardial dysfunction.MethodsUltrasound, Doppler examination and echocardiography was applied to classify cases and controls. Amniotic fluid nt-proBNP to amniotic fluid total protein ratio was calculated and compared to the gestational age-dependent reference intervals. In a subset of cases, fetal and maternal plasma nt-proBNP levels were determined.ResultsSpecimen from 391 fetuses could be analyzed (171 cases, 220 controls). There was a high correlation between amniotic fluid and fetal blood nt-proBNP levels (r = 0.441 for cases; r = 0.515 for controls), whereas no correlation could be detected between maternal and fetal (blood and amniotic fluid) nt-proBNP concentrations. Specificity and positive likelihood ratio of amniotic fluid nt-proBNP to amniotic fluid total protein ratio were high (0.97 and 4.3, respectively).ConclusionAmniotic fluid nt-proBNP measurement allows diagnostic confirmation of fetal myocardial dysfunction. It may serve as a useful adjunct in addition and correlation to existing tests of myocardial function, particularly in the context of invasive fetal therapy, where access to the amniotic cavity is part of the procedure.

Age-dependent reference intervals for estimated and measured glomerular filtration rate.

BackgroundDefining mean and reference intervals for glomerular filtration rate (GFR) has been the subject of only a limited number of studies and review articles, with contradicting statements about the mean. Normal measured GFR (mGFR) values of ∼120–130 mL/min/1.73 m2 have long been the referenced values for young adults but seem to be too high according to recent studies. Reference intervals are difficult to define because of the age decline of GFR, which is also observed in healthy subjects. Little data are available for subjects >70 years of age.MethodsBased on the reference intervals for serum creatinine (SCr) and the recently published full-age spectrum (FAS) equation, we define simple age-dependent equations for the reference limits of GFR. The mGFR of 633 living potential kidney donors was used to validate the new formulae that define the reference interval.ResultsThe reference limits for estimated GFR (eGFR), calculated by entering the reference limits for SCr into the FAS equation closely correspond with published reference limits for mGFR. Of the mGFRs of potential living kidney donors, 97.2% lie between the newly defined reference limits for GFR.ConclusionSCr reference limits may serve to define age-dependent reference limits for eGFR and mGFR.

Preanalytical validation and reference values for a mass spectrometric assay of serum vanillylmandelic acid for screening of catecholamine secreting neuroendocrine tumors

BackgroundUrinary vanillylmandelic acid (VMA) is used to diagnose and monitor catecholamine secreting neuroendocrine tumors (NETs). We developed and validated a new liquid chromatography tandem mass spectrometry (LC–MS/MS) assay for determination of serum VMA. MethodsWe used serum samples from healthy volunteers (n=314) and patients suspected for NET (n=36). Deuterated VMA as an internal standard was added to samples before solid phase extraction (SPE) and LC–MS/MS analysis. We studied the effects of sample storage, sampling device and a meal on serum VMA and metanephrine concentrations. Diurnal variation and age-dependent reference intervals were established. The diagnostic performance was compared with a urinary HPLC assay for VMA and metanephrines and a serum metanephrine LC–MS/MS assay. ResultsSerum VMA is stable at least for one day at +4°C, seven days at room temperature and 98days at −20°C. Type of sampling device was not critical, but elevated serum VMA occurs after a meal (p=0.031). Serum VMA increased with age. Therefore, we suggest clinical cut-off values of 62nmol/L, 80nmol/L and 108nmol/L for age groups 18–50yrs, 51–70yrs and >70yrs, respectively. Comparison between a urinary VMA HPLC assay and serum VMA LC–MS/MS assay showed good correlation. ConclusionsOur LC–MS/MS assay is fast and sensitive and suits well for use in a clinical laboratory. Compared to 24-h urine collection our serum assay enables well controlled sampling and convenient preanalytical steps.

Effect of age and gender on reference intervals of red blood cell distribution width (RDW) and mean red cell volume (MCV).

Red blood cell distribution width (RDW) was recently shown to be age-dependent when using Sysmex XE-2100 hematology analyzers. As measuring RDW is subject to technology, we have investigated if this relation also exists when using a different hematology analyzer, Abbott CELL-DYN Sapphire. In addition, as RDW is generally expressed relative to mean red blood cell volume (MCV), we have explored how MCV influences the age-dependency of RDW. We measured RDW and MCV in a large cohort and calculated RDW-SD (the "absolute" RDW), which does not contain MCV. For establishing reference intervals we used Bhattacharya statistics. In our study cohort of 8089 individuals we found a strong association between RDW and age, but not with gender. Also MCV showed an age-related increase over the entire age range. The conventional RDW increased by 6% from the youngest to oldest age class, whereas RDW-SD increased by nearly 15%. This difference was caused by a mean age-related increase in MCV of 6.6%. Age-dependent reference intervals were established for RDW, RDW-SD and MCV. The age-dependency of RDW seems to be a universal biological feature rather than related with a single type of hematology analyzer. As not only RDW, but also MCV increases with age, we propose that future studies on the prognostic significance of RDW take its age-dependency into account and focus on RDW-SD as a potential marker of adverse events in many clinical conditions.

Reference intervals for plasma pro-gastrin releasing peptide (ProGRP) levels in healthy adults of Chinese Han ethnicity.

To determine the upper limits of the 95th percentile reference intervals (RIs) for the detection of the pro-gastrin releasing peptide (ProGRP) in plasma according to the Clinical and Laboratory Standards Institute (CLSI) C28-A3 guideline in a population of adult Chinese of Han ethnicity. Plasma samples were collected from 578 healthy adults. Plasma ProGRP values were measured by chemiluminescent microparticle immunoassay (CMIA) on Abbot ARCHITECT i2000 system analyzers. Plasma ProGRP values did not conform to a Gaussian distribution, and thus non-parametric statistical methods were used to calculate RIs. Plasma ProGRP levels significantly increased with age, thus age-dependent RIs were determined. The upper limit of the 95th percentile RIs for plasma ProGRP was ≤57.26 pg/mL for young adults (18-50 years) and ≤81.42 pg/mL for old adults (&gt;50 years). By CMIA, we established distinct age-dependent RIs for plasma ProGRP in healthy adults of Chinese Han ethnicity, thus generating a valuable reference for future clinical and laboratory studies.

Pediatric reference intervals for serum copper and zinc

BackgroundCopper and zinc are essential trace elements and nutrients, which have important roles in physiology and disease and as cofactors in many metabolic pathways. We established age- and gender-specific reference intervals for serum copper and zinc, with a large healthy pediatric population. MethodsA total of 2115 clinically healthy children, 85.4% Caucasian, were enrolled: 1098 boys and 1017 girls 0.5–18y. Serum copper and zinc concentrations were measured by inductively coupled plasma-mass spectrometry (analytical measurement range: 10–400μg/dl for each). Statistical analyses and related calculations were performed using the STATA Statistical software and R software. ResultsThere were no significant effects of gender or fasting status. For copper, but not zinc, there were significant differences in the reference intervals by age. Based on the central 95% and 95% confidence intervals, the resulting reference interval determined for serum zinc was 64–124μg/dl. The age-dependent reference intervals for serum copper were 75–153μg/dl for those <10.3y, 64–132μg/dl for those 10.3–12.5y and 57–129μg/dl for those >12.5y. ConclusionsOur data defined reference intervals for serum copper and zinc in an American, clinically healthy, pediatric population.

Reference Intervals for Serum Alpha-Fetoprotein and Carcinoembryonic Antigen in Chinese Han Ethnic Males from the Fangchenggang Area Male Health and Examination Survey

We calculated upper 95% reference limits for serum alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) according to the CLSI/NCCLS C28-A3 guideline. Serum samples from 1400 healthy male subjects were collected from the Fangchenggang Area Male Health and Examination Survey (FAMHES). Serum AFP and CEA values were measured by electrochemiluminescence immunoassay on COBAS 6000 system E601 (Elecsys module) immunoassay analyzers. Serum AFP and CEA values were not normally distributed but log normally distributed. The upper 95% reference limits of the reference intervals were ≤ 4.76 IU/mL (nonparametric) or ≤ 4.56 IU/mL (parametric) for AFP and ≤ 5.57 ng/mL (nonparametric) or ≤ 5.82 ng/mL (parametric) for CEA. The distribution of AFP levels was found to be consistent between the non-smoking and smoking (p=0.740) and non-drinking and drinking groups (p=0.698). The distribution of serum CEA levels was significantly higher in the smoking than the non-smoking group (p<10⁵), whereas there was no significant difference in this respect between the non-drinking and drinking groups (p=0.147). A significant increase with age was found both for serum AFP and CEA levels, and the age-dependent reference intervals were calculated. The reference intervals for serum AFP and CEA show a slight deviation compared to previously reported reference levels. Distinct reference intervals of serum CEA must be established for smoking and non-smoking populations. In addition, age-dependent reference intervals should be implemented in clinical laboratories.

IgG Subclass Concentrations in Certified Reference Material 470 and Reference Values for Children and Adults Determined with The Binding Site Reagents

There is currently no international reference preparation for IgG subclass (IgGSc) quantification. This situation has led to calibration differences among assays and a variety of reference interval values with consequential difficulties in comparing results. We therefore evaluated IgGSc concentrations in Certified Reference Material 470 (CRM 470). Pure, polyclonal IgG1, -2, -3, and -4 were prepared from a large serum pool for use as primary standards. The IgG mass in each preparation was calculated from amino-acid analysis data. IgGSc concentrations were assessed in CRM 470 by nephelometry with modern analytical techniques, using these reference preparations. Subsequently, IgGSc concentrations were measured in 380 healthy individuals (250 males and 130 females), and age-dependent reference intervals were established. IgGSc concentrations in CRM 470 were as follows: IgG1, 5028 mg/L; IgG2, 3418 mg/L; IgG3, 579 mg/L, and IgG4, 381 mg/L, with a total IgG concentration of 9406 mg/L, 2.83% below the certified total IgG value of 9680 mg/L. Age-dependent percentile curves for the four IgGSc were constructed using a Box-Cox transformation. Maximum median values were as follows: IgG1, 6.02 g/L at 11 years; IgG2, 3.45 g/L at 31 years; IgG3, 0.63 g/L at 17 years; and IgG4, 0.48 g/L at 14 years. No significant sex-related differences were observed. The correlation between the summation of individual IgGSc and separate measurements of total IgG concentrations was good and supports the accuracy of the results. The results are based on The Binding Site assays and should not be considered appropriate for other assays unless so demonstrated.

Alpha-fetoprotein in plasma and serum of healthy adults: preanalytical, analytical and biological sources of variation and construction of age-dependent reference intervals

Alpha-fetoprotein (AFP) is a tumor marker for hepatomas and germ cell tumors, and the serum concentration has prognostic significance in other diseases. We examined the normal serum concentration of AFP in adults and sources of variation in the immunochemical variation of AFP. The serum concentration of the tumor marker alpha-fetoprotein (S-AFP) was log-normally distributed in 284 adult blood donors. S-AFP increased with age (p<10 7), whereas no gender-related difference was found. Reference intervals (95-interpercentile) were constructed for persons 40 years (0.60-9.30kIU/L) and >40 years (1.40-12.60kIU/L). The concentration of AFP was significantly, albeit slightly, higher in serum than in plasma, whereas hemolysis, pretreatment with KCl and food intake did not influence S-AFP. S-AFP only changed 6% when measured twice 2 months apart (p=0.04). Three enzyme immunoassays, using three different anti-AFP monoclonal antibodies for detection, were compared and two assays gave S-AFP values significantly higher, 2.8% (p=0.03) and 19.0% (p<10 4), than the other assay. Thus, the choice of antibody may influence the result of immunochemical concentration determination. This can b eexplained by the existence of conformational variants of AFP with different antibody reactivities, and calls for careful standardization of monoclonal antibodies used in assays for AFP. With broad population reference ranges and slight intra-personal variation, the most effective reference range for S-AFP is previous values obtained in the same person.