A pipeline for the fully automated estimation of continuous reference intervals using real-world data.

Reference intervals are essential for interpreting laboratory test results. Continuous reference intervals precisely capture physiological age-specific dynamics that occur throughout life, and thus have the potential to improve clinical decision-making. However, established approaches for estimating continuous reference intervals require samples from healthy individuals, and are therefore substantially restricted. Indirect methods operating on routine measurements enable the estimation of one-dimensional reference intervals, however, no automated approach exists that integrates the dependency on a continuous covariate like age. We propose an integrated pipeline for the fully automated estimation of continuous reference intervals expressed as a generalized additive model for location, scale and shape based on discrete model estimates using an indirect method (refineR). The results are free of subjective user-input, enable conversion of test results into z-scores and can be integrated into laboratory information systems. Comparison of our results to established and validated reference intervals from the CALIPER and PEDREF studies and manufacturers' package inserts shows good agreement of reference limits, indicating that the proposed pipeline generates high-quality results. In conclusion, the developed pipeline enables the generation of high-precision percentile charts and continuous reference intervals. It represents the first parameter-less and fully automated solution for the indirect estimation of continuous reference intervals.

Estimation of Reference Intervals from Routine Data Using the refineR Algorithm-A Practical Guide.

BACKGROUND: Accurate reference intervals are essential for the interpretation of laboratory test results. Typically, they are determined by the central 95% range of test results from a predefined reference population. As these direct studies can face practical and ethical challenges, indirect methods using routine measurements offer an alternative approach. METHODS: We provide step-by-step guidance on how to apply an indirect method in practice using refineR, the most recently published indirect method, and showcase the application by evaluating real-world data of 12 prespecified analytes. Measurements were retrieved from ARUP Laboratories' data warehouse, and were obtained from routine patient testing on cobas c502 or e602 analyzers. Test results were prefiltered and cleaned and, if necessary, physiologically partitioned prior to estimating reference intervals using refineR. Estimated reference intervals were then compared to established intervals provided by the manufacturer. RESULTS: For most analytes, the reference intervals estimated by refineR were comparable to those provided by the manufacturer, shown by overlapping confidence intervals at both reference limits, or only the upper or lower limit. For thyroid-stimulating hormone, refineR estimated higher reference limits, while estimates for prealbumin were lower compared to the established reference interval. CONCLUSIONS: We applied the refineR algorithm to a variety of real-world data sets resulting in reference intervals similar to intervals previously established by direct methods. We further provide practical guidance and a code example on how to apply an indirect method in a real-world scenario facilitating their access and thus their use in laboratory settings.

RIbench: A Proposed Benchmark for the Standardized Evaluation of Indirect Methods for Reference Interval Estimation.

BACKGROUND: Indirect methods leverage real-world data for the estimation of reference intervals. These constitute an active field of research, and several methods have been developed recently. So far, no standardized tool for evaluation and comparison of indirect methods exists. METHODS: We provide RIbench, a benchmarking suite for quantitative evaluation of any existing or novel indirect method. The benchmark contains simulated test sets for 10 biomarkers mimicking routine measurements of a mixed distribution of non-pathological (reference) values and pathological values. The non-pathological distributions represent 4 common distribution types: normal, skewed, heavily skewed, and skewed-and-shifted. To identify strengths and weaknesses of indirect methods, test sets have varying sample sizes and pathological distributions differ in location, extent of overlap, and fraction. For performance evaluation, we use an overall benchmark score and sub-scores derived from absolute z-score deviations between estimated and true reference limits. We illustrate the application of RIbench by evaluating and comparing the Hoffmann method and 4 modern indirect methods -TML (Truncated-Maximum-Likelihood), kosmic, TMC (Truncated-Minimum-Chi-Square), and refineR- against one another and against a nonparametric direct method (n = 120). RESULTS: For the modern indirect methods, pathological fraction and sample size had a strong influence on the results: With a pathological fraction up to 20% and a minimum sample size of 5000, most methods achieved results comparable or superior to the direct method. CONCLUSIONS: We present RIbench, an open-source R-package, for the systematic evaluation of existing and novel indirect methods. RIbench can serve as a tool for enhancement of indirect methods, improving the estimation of reference intervals.

refineR: A Novel Algorithm for Reference Interval Estimation from Real-World Data.

Reference intervals are essential for the interpretation of laboratory test results in medicine. We propose a novel indirect approach to estimate reference intervals from real-world data as an alternative to direct methods, which require samples from healthy individuals. The presented refineR algorithm separates the non-pathological distribution from the pathological distribution of observed test results using an inverse approach and identifies the model that best explains the non-pathological distribution. To evaluate its performance, we simulated test results from six common laboratory analytes with a varying location and fraction of pathological test results. Estimated reference intervals were compared to the ground truth, an alternative indirect method (kosmic), and the direct method (N = 120 and N = 400 samples). Overall, refineR achieved the lowest mean percentage error of all methods (2.77%). Analyzing the amount of reference intervals within ± 1 total error deviation from the ground truth, refineR (82.5%) was inferior to the direct method with N = 400 samples (90.1%), but outperformed kosmic (70.8%) and the direct method with N = 120 (67.4%). Additionally, reference intervals estimated from pediatric data were comparable to published direct method studies. In conclusion, the refineR algorithm enables precise estimation of reference intervals from real-world data and represents a viable complement to the direct method.

A Multicenter Evaluation of a Point-of-Care Blood Glucose Meter System in Critically Ill Patients.

BACKGROUND: Our purpose was to evaluate the performance of the ACCU-CHEK® Inform II blood glucose monitoring system (Roche Diagnostics GmbH) compared with the perchloric acid hexokinase (PCA-HK) comparator method on the cobas® 6000 analyzer (Roche Diagnostics International Ltd) in critically ill patients. METHODS: Overall, 476 arterial (376 pediatric/adult, 100 neonate), 375 venous, and 100 neonatal heel-stick whole-blood samples were collected and evaluated from critical care settings at 10 US hospitals, including the emergency department, medical and surgical intensive care units (ICUs), and neonatal and pediatric ICUs. The ACCU-CHEK Inform II system was evaluated at 2 cutoff boundaries: boundary 1 was ≥95% of results within ±12 mg/dL of the reference (samples with blood glucose <75 mg/dL) or ±12% of the reference (glucose ≥75 mg/dL), and boundary 2 was ≥98% of results within ±15 mg/dL or ±15% of the reference. Clinical performance was assessed by evaluating sample data using Parkes error grid, Monte Carlo simulation, and sensitivity and specificity analyses to estimate clinical accuracy and implications for insulin dosing when using the ACCU-CHEK Inform II system. RESULTS: Proportions of results within evaluation boundaries 1 and 2, respectively, were 96% and 98% for venous samples, 94% and 97% for pediatric and adult arterial samples, 84% and 98% for neonatal arterial samples, and 96% and 100% for neonatal heel-stick samples. Clinical evaluation demonstrated high specificity and sensitivity, with low risk of potential insulin-dosing errors. CONCLUSIONS: The ACCU-CHEK Inform II system demonstrated clinically acceptable performance against the PCA-HK reference method for blood glucose monitoring in a diverse population of critically ill patients in US care settings.

Clinical risk assessment of biotin interference with a high-sensitivity cardiac troponin T assay.

Objectives Biotin >20.0 ng/mL (81.8 nmol/L) can reduce Elecsys® Troponin T Gen 5 (TnT Gen 5; Roche Diagnostics) assay recovery, potentially leading to false-negative results in patients with suspected acute myocardial infarction (AMI). We aimed to determine the prevalence of elevated biotin and AMI misclassification risk from biotin interference with the TnT Gen 5 assay. Methods Biotin was measured using an Elecsys assay in two cohorts: (i) 797 0-h and 646 3-h samples from 850 US emergency department patients with suspected acute coronary syndrome (ACS); (ii) 2023 random samples from a US laboratory network, in which biotin distributions were extrapolated for higher values using pharmacokinetic modeling. Biotin >20.0 ng/mL (81.8 nmol/L) prevalence and biotin 99th percentile values were calculated. AMI misclassification risk due to biotin interference with the TnT Gen 5 assay was modeled using different assay cutoffs and test timepoints. Results ACS cohort: 1/797 (0.13%) 0-h and 1/646 (0.15%) 3-h samples had biotin >20.0 ng/mL (81.8 nmol/L); 99th percentile biotin was 2.62 ng/mL (10.7 nmol/L; 0-h) and 2.38 ng/mL (9.74 nmol/L; 3-h). Using conservative assumptions, the likelihood of false-negative AMI prediction due to biotin interference was 0.026% (0-h result; 19 ng/L TnT Gen 5 assay cutoff). US laboratory cohort: 15/2023 (0.74%) samples had biotin >20.0 ng/mL (81.8 nmol/L); 99th percentile biotin was 16.6 ng/mL (68.0 nmol/L). Misclassification risk due to biotin interference (19 ng/L TnT Gen 5 assay cutoff) was 0.025% (0-h), 0.0064% (1-h), 0.00048% (3-h), and <0.00001% (6-h). Conclusions Biotin interference has minimal impact on the TnT Gen 5 assay's clinical utility, and the likelihood of false-negative AMI prediction is extremely low.

Comparative transcriptome profiling of maize coleoptilar nodes during shoot-borne root initiation.

Maize (Zea mays) develops an extensive shoot-borne root system to secure water and nutrient uptake and to provide anchorage in the soil. In this study, early coleoptilar node (first shoot node) development was subjected to a detailed morphological and histological analysis. Subsequently, microarray profiling via hybridization of oligonucleotide microarrays representing transcripts of 31,355 unique maize genes at three early stages of coleoptilar node development was performed. These pairwise comparisons of wild-type versus mutant rootless concerning crown and seminal roots (rtcs) coleoptilar nodes that do not initiate shoot-borne roots revealed 828 unique transcripts that displayed RTCS-dependent expression. A stage-specific functional analysis revealed overrepresentation of "cell wall," "stress," and "development"-related transcripts among the differentially expressed genes. Differential expression of a subset of 15 of 828 genes identified by these microarray experiments was independently confirmed by quantitative real-time-polymerase chain reaction. In silico promoter analyses revealed that 100 differentially expressed genes contained at least one LATERAL ORGAN BOUNDARIES domain (LBD) motif within 1 kb upstream of the ATG start codon. Electrophoretic mobility shift assay experiments demonstrated RTCS binding for four of these promoter sequences, supporting the notion that differentially accumulated genes containing LBD motifs are likely direct downstream targets of RTCS.

Influence of land-use intensity on the spatial distribution of N-cycling microorganisms in grassland soils.

A geostatistical approach using replicated grassland sites (10 m × 10 m) was applied to investigate the influence of grassland management, i.e. unfertilized pastures and fertilized mown meadows representing low and high land-use intensity (LUI), on soil biogeochemical properties and spatial distributions of ammonia-oxidizing and denitrifying microorganisms in soil. Spatial autocorrelations of the different N-cycling communities ranged between 1.4 and 7.6 m for ammonia oxidizers and from 0.3 m for nosZ-type denitrifiers to scales >14 m for nirK-type denitrifiers. The spatial heterogeneity of ammonia oxidizers and nirS-type denitrifiers increased in high LUI, but decreased for biogeochemical properties, suggesting that biotic and/or abiotic factors other than those measured are driving the spatial distribution of these microorganisms at the plot scale. Furthermore, ammonia oxidizers (amoA ammonia-oxidizing archaea and amoA ammonia-oxidizing bacteria) and nitrate reducers (napA and narG) showed spatial coexistence, whereas niche partitioning was found between nirK- and nirS-type denitrifiers. Together, our results indicate that spatial analysis is a useful tool to characterize the distribution of different functional microbial guilds with respect to soil biogeochemical properties and land-use management. In addition, spatial analyses allowed us to identify distinct distribution ranges indicating the coexistence or niche partitioning of N-cycling communities in grassland soil.

Nonadditive protein accumulation patterns in Maize (Zea mays L.) hybrids during embryo development.

Heterosis describes the superior performance of heterozygous F(1)-hybrid plants compared to their homozygous parental inbred lines. In the present study, heterosis was detected for length, weight, and the time point of seminal root primordia initiation in maize (Zea mays L.) embryos of the reciprocal F(1)-hybrids UH005xUH250 and UH250xUH005. A two-dimensional gel electrophoresis (2-DE) proteome survey of the most abundant proteins of the reciprocal hybrids and their parental inbred lines 25 and 35 days after pollination revealed that 141 of 597 detected proteins (24%) exhibited nonadditive accumulation in at least one hybrid. Approximately 44% of all nonadditively accumulated proteins displayed an expression pattern that was not distinguishable from the low parent value. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) analyses and subsequent functional classification of the 141 proteins revealed that development, protein metabolism, redox-regulation, glycolysis, and amino acid metabolism were the most prominent functional classes among nonadditively accumulated proteins. In 35-day-old embryos of the hybrid UH250xUH005, a significant up-regulation of enzymes related to glucose metabolism which often exceeded the best parent values was observed. A comparison of nonadditive protein accumulation between rice and maize embryo data sets revealed a significant overlap of nonadditively accumulated proteins suggesting conserved organ- or tissue-specific regulatory mechanisms in monocots related to heterosis.

Regulation of the maize (Zea mays L.) embryo proteome by RTCS which controls seminal root initiation.

Seminal roots are initiated at the scutellar node during maize (Zea mays L.) embryo development. The maize mutant rtcs (rootless concerning crown and seminal roots) does not initiate seminal roots while its wild-type siblings form on average 2.9 seminal roots per seedling. In this study, proteome profiles of 25-day-old immature maize embryos were compared between wild-type and rtcs plants via two-dimensional electrophoresis (2-DE). Electrospray ionization tandem mass spectrometry (ESI-MS/MS) identified 23 proteins encoded by 21 different genes that were differentially accumulated between wild-type and rtcs embryos (Fc> or =2; FDR<10%). Among the differentially accumulated proteins, two isoforms of a phosphoglycerate kinase and a malate dehydrogenase were preferentially accumulated in wild-type embryos. Both enzymes are related to the generation of energy-rich ATP or NADPH molecules and are crucial checkpoints of cellular energetics in plants. Comparison of embryonic proteins differentially accumulated between wild-type and rtcs embryos revealed little overlap with proteins differentially accumulated between wild-type and rum1 embryos which also do not initiate seminal roots. This might be due to distinct influences of RTCS and RUM1 on the composition of the embryo proteome, but could also be explained by different stages of embryo development that were analyzed in these studies.

Background correction of two-colour cDNA microarray data using spatial smoothing methods.

The analysis of two-colour cDNA microarray data usually involves subtracting background values from foreground values prior to normalization and further analysis. This approach has the advantage of reducing bias and the disadvantage of blowing up the variance of lower abundant spots. Whenever background subtraction is considered, it implicitly assumes locally constant background values. In practice, this assumption is often not met, which casts doubts on the usefulness of simple background subtraction. In order to improve background correction, we propose local background smoothing within the pre-processing pipeline of cDNA microarray data prior to background correction. For this purpose, we employ a geostatistical framework with ordinary kriging using both isotropic and anisotropic models of spatial correlation and 2-D locally weighted regression. We show that application of local background smoothing prior to background correction is beneficial in comparison to using raw background estimates. This is done using data of a self-versus-self experiment in Arabidopsis where subsets of differentially expressed genes were simulated. Using locally smoothed background values in conjunction with existing background correction methods increases the power, increases the accuracy and decreases the number of false positive results.

Specification of cortical parenchyma and stele of maize primary roots by asymmetric levels of auxin, cytokinin, and cytokinin-regulated proteins.

In transverse orientation, maize (Zea mays) roots are composed of a central stele that is embedded in multiple layers of cortical parenchyma. The stele functions in the transport of water, nutrients, and photosynthates, while the cortical parenchyma fulfills metabolic functions that are not very well characterized. To better understand the molecular functions of these root tissues, protein- and phytohormone-profiling experiments were conducted. Two-dimensional gel electrophoresis combined with electrospray ionization tandem mass spectrometry identified 59 proteins that were preferentially accumulated in the cortical parenchyma and 11 stele-specific proteins. Hormone profiling revealed preferential accumulation of indole acetic acid and its conjugate indole acetic acid-aspartate in the stele and predominant localization of the cytokinin cis-zeatin, its precursor cis-zeatin riboside, and its conjugate cis-zeatin O-glucoside in the cortical parenchyma. A root-specific beta-glucosidase that functions in the hydrolysis of cis-zeatin O-glucoside was preferentially accumulated in the cortical parenchyma. Similarly, four enzymes involved in ammonium assimilation that are regulated by cytokinin were preferentially accumulated in the cortical parenchyma. The antagonistic distribution of auxin and cytokinin in the stele and cortical parenchyma, together with the cortical parenchyma-specific accumulation of cytokinin-regulated proteins, suggest a molecular framework that specifies the function of these root tissues that also play a role in the formation of lateral roots from pericycle and endodermis cells.

Heterotic patterns of sugar and amino acid components in developing maize kernels.

Heterosis is the superior performance of hybrids over their inbred parents. Despite its importance, little is known about the genetic and molecular basis of this phenomenon. Heterosis has been extensively exploited in plant breeding, particularly in maize (Zea mays, L.), and is well documented in the B73 and Mo17 maize inbred lines and their F1 hybrids. In this study, we determined the dry matter, the levels of starch and protein components and a total of 24 low-molecular weight metabolites including sugars, sugar-phosphates, and free amino acids, in developing maize kernels between 8 and 30 days post-pollination (DPP) of the hybrid B73 x Mo17 and its parental lines. The tissue specificity of amino acid and protein content was investigated between 16 and 30 DPP. Key observations include: (1) most of the significant differences in the investigated tissue types occurred between Mo17 and the other two genotypes; (2) heterosis of dry matter and metabolite content was detectable from the early phase of kernel development onwards; (3) the majority of metabolites exhibited an additive pattern. Nearly 10% of the metabolites exhibited nonadditive effects such as overdominance, underdominance, and high-parent and low-parent dominance; (4) The metabolite composition was remarkably dependent on kernel age, and this large developmental effect could possibly mask genotypic differences; (5) the metabolite profiles and the heterotic patterns are specific for endosperm and embryo. Our findings illustrate the power of metabolomics to characterize heterotic maize lines and suggest that the metabolite composition is a potential marker in the context of heterosis research.

Tissue specific control of the maize (Zea mays L.) embryo, cortical parenchyma, and stele proteomes by RUM1 which regulates seminal and lateral root initiation.

The different root types of maize (Zea mays L.) originate from distinct tissues during development. The maize mutant rum1 (rootless with undetectable meristems 1) does not initiate seminal roots and lateral roots in the primary root. While seminal roots are laid down during embryogenesis, endodermis cells of the parenchyma, and pericycle cells of the stele contribute to the postembryonic initiation of lateral roots. In this study, tissue specific protein profiles of immature embryo, cortical parenchyma which includes endodermis, cortex and epidermis cell layers, and stele tissues were compared between wild-type and rum1 via 2-DE. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) identified 86 proteins encoded by 69 genes that were differentially accumulated between wild-type and rum1 (Fc>or=2; FDR<10%) demonstrating that RUM1 affects the proteome composition of cortical parenchyma, stele and embryo tissues. While several protein isoforms, protein families or members of biochemical pathways regulated by RUM1 were differentially accumulated in at least two tissues, other proteins displayed tissue specific expression differences. Multiple members of the globulin gene family displayed, for example, embryo specific expression differences, while different glycolysis related enzymes were differentially expressed in all three analyzed tissues. Proteins related to signal transduction and cell fate were overrepresented in cortical parenchyma versus embryo and embryo versus stele tissues, respectively, and might imply tissue specific functions of these protein classes.