Reliable biological and multi-omics research through biometrology.

Metrology is the science of measurement and its applications, whereas biometrology is the science of biological measurement and its applications. Biometrology aims to achieve accuracy and consistency of biological measurements by focusing on the development of metrological traceability, biological reference measurement procedures, and reference materials. Irreproducibility of biological and multi-omics research results from different laboratories, platforms, and analysis methods is hampering the translation of research into clinical uses and can often be attributed to the lack of biologists' attention to the general principles of metrology. In this paper, the progresses of biometrology including metrology on nucleic acid, protein, and cell measurements and its impacts on the improvement of reliability and comparability in biological research are reviewed. Challenges in obtaining more reliable biological and multi-omics measurements due to the lack of primary reference measurement procedures and new standards for biological reference materials faced by biometrology are discussed. In the future, in addition to establishing reliable reference measurement procedures, developing reference materials from single or multiple parameters to multi-omics scale should be emphasized. Thinking in way of biometrology is warranted for facilitating the translation of high-throughput omics research into clinical practices.

Implementing metrological traceability of C-reactive protein measurements: consensus summary from the Joint Committee for Traceability in Laboratory Medicine Workshop.

The Joint Committee for Traceability in Laboratory Medicine (JCTLM) currently lists the secondary commutable certified reference material (CRM) ERM DA-474/IFCC (DA-474) "C-Reactive Protein in Human Serum" and two generic immunoassay-based method principles as the basis for implementing the metrological traceability of C-reactive protein (CRP) measurements by end-user measurement procedures used by medical laboratories. The current metrological traceability has produced well harmonized results for clinical samples among different end-user measurement procedures. New higher-order pure substance and secondary commutable CRMs have been nominated for listing by the JCTLM. However, the data supporting performance of these new candidate CRMs, including use of new mass spectrometry based candidate reference measurement procedures (RMPs), was not clear regarding the influence that introducing these new CRMs would have on the current well harmonized results achieved with the existing metrological traceability to DA-474. The clinically relevant CRP measurand in blood serum or plasma is a pentamer of identical subunits, which adds complexity to the application of higher-order CRMs and RMPs. The JCTLM convened a workshop in December 2022 to review the appropriate implementation of metrological traceability of CRP measurements. The workshop consensus was that the extent-of-equivalence data must include considerations about the impact of a new CRM when used for its intended purpose in the calibration hierarchies of existing end-user measuring systems; and that a new RMP must compare results with another existing well validated candidate RMP or with a globally available end-user measurement system.

Overcoming challenges regarding reference materials and regulations that influence global standardization of medical laboratory testing results.

BACKGROUND: Standardized results for laboratory tests are particularly important when their interpretation depends on fixed medical practice guidelines or common reference intervals. The medical laboratory community has developed a roadmap for an infrastructure to achieve standardized test results described in the International Organization for Standardization standard 17511:2020 In vitro diagnostic medical devices - Requirements for establishing metrological traceability of values assigned to calibrators, trueness control materials and human samples. Among the challenges to implementing metrological traceability are the availability of fit-for-purpose matrix-based certified reference materials (CRMs) and requirements for regulatory review that differ among countries. A workshop in December 2021 focused on these two challenges and developed recommendations for improved practices. DISCUSSION: The participants agreed that prioritization of measurands for standardization should be based on their impact on medical decisions in a clinical pathway. Ensuring that matrix-based CRMs are globally available for more measurands will enable fit-for-purpose calibration hierarchies for more laboratory tests. Regulation of laboratory tests is important to ensure safety and effectiveness for the populations served. Because regulations are country or region specific, manufacturers must submit recalibration changes intended to standardize results for regulatory review to all areas in which a measuring system is marketed. RECOMMENDATIONS: A standardization initiative requires collaboration and planning among all interested stakeholders. Global collaboration should be further developed for prioritization of measurands for standardization, and for coordinating the production and supply of CRMs worldwide. More uniform regulatory submission requirements are desirable when recalibration is implemented to achieve internationally standardized results.

Metrological framework to support accurate, reliable, and reproducible nucleic acid measurements.

Nucleic acid analysis is used in many areas of life sciences such as medicine, food safety, and environmental monitoring. Accurate, reliable measurements of nucleic acids are crucial for maximum impact, yet users are often unaware of the global metrological infrastructure that exists to support these measurements. In this work, we describe international efforts to improve nucleic acid analysis, with a focus on the Nucleic Acid Analysis Working Group (NAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM). The NAWG is an international group dedicated to improving the global comparability of nucleic acid measurements; its primary focus is to support the development and maintenance of measurement capabilities and the dissemination of measurement services from its members: the National Metrology Institutes (NMIs) and Designated Institutes (DIs). These NMIs and DIs provide DNA and RNA measurement services developed in response to the needs of their stakeholders. The NAWG members have conducted cutting edge work over the last 20 years, demonstrating the ability to support the reliability, comparability, and traceability of nucleic acid measurement results in a variety of sectors.

Anisotropic confinement of chromophores induces second-order nonlinear optics in a nanoporous photonic metamaterial.

Second-order nonlinear optics is the base for a large variety of devices aimed at the active manipulation of light. However, physical principles restrict its occurrence to non-centrosymmetric, anisotropic matter. This significantly limits the number of base materials exhibiting nonlinear optics. Here, we show that embedding chromophores in an array of conical channels 13 nm across in monolithic silica results in mesoscopic anisotropic matter and thus in a hybrid material showing second-harmonic generation. This nonlinear optics is compared to the one achieved in corona-poled polymer films containing the identical chromophores. It originates in the confinement-induced orientational order of the elongated guest molecules in the nanochannels. This leads to a non-centrosymmetric dipolar order and hence to a nonlinear light-matter interaction on the sub-wavelength, single-pore scale. Our study demonstrates that the advent of large-scale, self-organized nanoporosity in monolithic solids along with the confinement-controllable orientational order of chromophores at the single-pore scale provides a reliable and accessible tool to design materials with a nonlinear meta-optics.

Structurally related peptide impurity identification and accurate quantification for synthetic oxytocin by liquid chromatography-high-resolution mass spectrometry.

Oxytocin (OXT) is an important peptide that is mainly used as a therapeutic drug to induce labor or strengthen uterine contractions, or to control bleeding after childbirth. OXT has also been reported as a biomarker linked to emotion, and as a potential biomarker for cancer diagnosis. The accurate purity characterization of OXT calibrators is critical for quality control of pharmaceuticals and the development of reference measurement systems for this analyte in laboratory medicine. OXT possesses the particular analytical measurement challenge of a disulfide bond. Accurate value assignment of the purity of oxytocin calibrators can be carried out by applying the mass balance approach or alternative approaches such as amino acid analysis, quantitative nuclear magnetic resonance spectrometry, and nitrogen determination. In order to avoid biases, all these approaches require a correction for structurally related peptide impurities. Structurally related peptide impurities present in a synthetic OXT material have been identified and quantified by a newly developed and in-house-validated liquid chromatography-high-resolution mass spectrometry (LC-hrMS) method. This method was adopted for the measurement of the study material used for an international comparison evaluating the competencies of laboratories to perform peptide characterization. Eighteen structurally related impurities were identified, confirmed, and accurately quantified in the OXT study material by using LC-hrMS. The study material contained a total mass fraction of 31.1 mg/g structurally related OXT impurities with an associated expanded uncertainty of 1.7 mg/g.

Identification and accurate quantification of structurally related peptide impurities in synthetic human C-peptide by liquid chromatography-high resolution mass spectrometry.

Peptides are an increasingly important group of biomarkers and pharmaceuticals. The accurate purity characterization of peptide calibrators is critical for the development of reference measurement systems for laboratory medicine and quality control of pharmaceuticals. The peptides used for these purposes are increasingly produced through peptide synthesis. Various approaches (for example mass balance, amino acid analysis, qNMR, and nitrogen determination) can be applied to accurately value assign the purity of peptide calibrators. However, all purity assessment approaches require a correction for structurally related peptide impurities in order to avoid biases. Liquid chromatography coupled to high resolution mass spectrometry (LC-hrMS) has become the key technique for the identification and accurate quantification of structurally related peptide impurities in intact peptide calibrator materials. In this study, LC-hrMS-based methods were developed and validated in-house for the identification and quantification of structurally related peptide impurities in a synthetic human C-peptide (hCP) material, which served as a study material for an international comparison looking at the competencies of laboratories to perform peptide purity mass fraction assignments. More than 65 impurities were identified, confirmed, and accurately quantified by using LC-hrMS. The total mass fraction of all structurally related peptide impurities in the hCP study material was estimated to be 83.3 mg/g with an associated expanded uncertainty of 3.0 mg/g (k = 2). The calibration hierarchy concept used for the quantification of individual impurities is described in detail. Graphical abstract ᅟ.

Calibration Strategies for FT-IR and Other Isotope Ratio Infrared Spectrometer Instruments for Accurate δ13C and δ18O Measurements of CO2 in Air.

This paper describes calibration strategies in laboratory conditions that can be applied to ensure accurate measurements of the isotopic composition of the CO2 in ultradry air, expressed as δ13C and δ18O on the VPDB scale, with either FT-IR (in this case a Vertex 70 V (Bruker)) or an isotope ratio infrared spectrometer (IRIS) (in this case a Delta Ray (Thermo Fisher Scientific)). In the case of FT-IR a novel methodology using only two standards of CO2 in air with different mole fractions but identical isotopic composition was demonstrated to be highly accurate for measurements of δ13C and δ18O with standard uncertainties of 0.09‰ and 1.03‰, respectively, at a nominal CO2 mole fraction of 400 µmol mol-1 in air. In the case of the IRIS system, we demonstrate that the use of two standards of CO2 in air of known but differing δ13C and δ18O isotopic composition allows standard uncertainties of 0.18‰ and 0.48‰ to be achieved for δ13C and δ18O measurements, respectively. The calibration strategies were validated using a set of five traceable primary reference gas mixtures. These standards, produced with whole air or synthetic air covered the mole fraction range of (378-420) µmol mol-1 and were prepared and/or value assigned either by the National Institute of Standards and Technology (NIST) or the National Physical Laboratory (NPL). The standards were prepared using pure CO2 obtained from different sources, namely, combustion; Northern Continental and Southern Oceanic Air and a gas well source, with δ13C values ranging between -35‰ and -1‰. The isotopic composition of all standards was value assigned at the Max Planck Institute for Biogeochemistry Jena (MPI-Jena).

Implementing a Reference Measurement System for C-Peptide: Successes and Lessons Learned.

BACKGROUND: Assessment of endogenous insulin secretion by measuring C-peptide concentrations is widely accepted. Recent studies have shown that preservation of even small amounts of endogenous C-peptide production in patients with type 1 diabetes reduces risks for diabetic complications. Harmonization of C-peptide results will facilitate comparison of data from different research studies and later among clinical laboratory results at different sites using different assay methods. CONTENT: This review provides an overview of the general process of harmonization and standardization and the challenges encountered with implementing a reference measurement system for C-peptide. SUMMARY: Efforts to harmonize C-peptide results are described, including those by the National Institute of Diabetes and Digestive and Kidney Diseases-led C-peptide Standardization Committee in the US, activities in Japan, efforts by the National Institute for Biological Standards and Control in the UK, as well as activities led by the Bureau International des Poids et Mesures and the National Metrology Institute in China. A traceability scheme is proposed along with the next steps for implementation. Suggestions are made for better collaboration to optimize the harmonization process for other measurands.

Impurity determination for hepcidin by liquid chromatography-high resolution and ion mobility mass spectrometry for the value assignment of candidate primary calibrators.

In metrology institutes, the state-of-the-art for purity analysis of peptides/proteins mainly addresses short and unfolded peptides. Important developments are anticipated for the characterization of nonlinear peptides or proteins. Hepcidin 1-25 is an interesting model system because this small protein contains four disulfide bridges with a particular connectivity that is difficult to reproduce and could induce a bias in quantification. Hepcidin 1-25 is involved in iron-related disorders and anemia, in an inflammatory context, and its clinical relevance in neurodegenerative disorders is under investigation. It is also an emerging biomarker. Recent inter-laboratory studies showed a need for standardization of hepcidin assay and the need to produce certified reference materials. This paper discusses two hepcidin standards from different synthesis pathways that have been characterized by high-resolution mass spectrometry and ion mobility mass spectrometry.

Ozone Cross-Section Measurement by Gas Phase Titration.

Elevated values of ground-level ozone damage health, vegetation, and building materials and are the subject of air quality regulations. Levels are monitored by networks using mostly ultraviolet (UV) absorption instruments, with traceability to standard reference photometers, relying on the UV absorption of ozone at the 253.65 nm line of mercury. We have redetermined the ozone cross-section at this wavelength based on gas phase titration (GPT) measurements. This is a well-known chemical method using the reaction of ozone (O3) with nitrogen monoxide (NO) resulting in nitrogen dioxide (NO2) and oxygen (O2). The BIPM GPT facility uses state-of-the-art flow measurement, chemiluminescence for NO concentration measurements, a cavity phase shift analyzer (CAPS) for NO2 measurements, and a UV ozone analyzer. The titration experiment is performed over the concentration range 100-500 nmol/mol, with NO and NO2 reactants/calibrants diluted down from standards with nominal mole fractions of 50 µmol/mol. Accurate measurements of NO, NO2, and O3 mole fractions allow the calculation of ozone absorption cross section values at 253.65 nm, and we report a value of 11.24 × 10-18 cm2 molecule-1 with a relative expanded uncertainty of 1.8% (coverage factor k = 2) based on nitrogen monoxide titration values and a value of 11.22 × 10-18 cm2 molecule-1 with a relative expanded uncertainty of 1.4% (coverage factor k = 2) based on nitrogen dioxide titration values. The excellent agreement between these values and recently published absorption cross-section measurements directly on pure ozone provide strong evidence for revising the conventionally accepted value of ozone cross section at 253.65 nm.

Methane standards made in whole and synthetic air compared by cavity ring down spectroscopy and gas chromatography with flame ionization detection for atmospheric monitoring applications.

There is evidence that the use of whole air versus synthetic air can bias measurement results when analyzing atmospheric samples for methane (CH4) and carbon dioxide (CO2). Gas chromatography with flame ionization detection (GC-FID) and wavelength scanned-cavity ring down spectroscopy (WS-CRDS) were used to compare CH4 standards produced with whole air or synthetic air as the matrix over the mole fraction range of 1600-2100 nmol mol(-1). GC-FID measurements were performed by including ratios to a stable control cylinder, obtaining a typical relative standard measurement uncertainty of 0.025%. CRDS measurements were performed using the same protocol and also with no interruption for a limited time period without use of a control cylinder, obtaining relative standard uncertainties of 0.031% and 0.015%, respectively. This measurement procedure was subsequently used for an international comparison, in which three pairs of whole air standards were compared with five pairs of synthetic air standards (two each from eight different laboratories). The variation from the reference value for the whole air standards was determined to be 2.07 nmol mol(-1) (average standard deviation) and that of synthetic air standards was 1.37 nmol mol(-1) (average standard deviation). All but one standard agreed with the reference value within the stated uncertainty. No significant difference in performance was observed between standards made from synthetic air or whole air, and the accuracy of both types of standards was limited only by the ability to measure trace CH4 levels in the matrix gases used to produce the standards.

Normal phase-liquid chromatography-tandem mass spectrometry with atmospheric pressure photoionization for the purity assessment of 17ß-estradiol.

A normal phase-liquid chromatography-hybrid tandem mass spectrometry (NP-LC-MS/MS) method utilizing atmospheric pressure photoionization (APPI) without dopant has been developed and implemented for the simultaneous determination of several estrogenic steroid hormones. The combination of both NP-LC and APPI-MS/MS tolerates the use of solvents that have the advantages of being self-doping for APPI and, at the same time, inhibit the in situ formation of estrogen dimers as frequently observed for conventional reversed phase (RP)-LC methods. The NP-LC-APPI-MS/MS method has been validated in-house, and its performance characteristics (linearity, repeatability, limits of detection, etc.) were assessed for use in the quantification of estrogens. Moreover, the method was used to characterize and determine the inherent related structure impurities in batches of ß-estradiol, required for the establishment of reference measurement systems for clinical chemistry and laboratory medicine, which served as candidate reference material for an organic purity assessment interlaboratory study (CCQM-K55.a) organized by the International Bureau of Weights and Measures (BIPM) Chemistry Department and carried out within the framework of the Organic Analysis Working Group (OAWG) of the Consultative Committee for Amount of Substance-Metrology in Chemistry (CCQM).

Mass balance method for the SI value assignment of the purity of organic compounds.

A mass balance method is described for determining the mass fraction of the main component of a high purity organic material. The resulting assigned value is established to be traceable to the SI and can be determined with a small associated measurement uncertainty. Pure organic materials with values and uncertainties determined in this way are necessary as primary calibrators of reference measurement systems in order to underpin the metrological traceability of routine measurement results. The method has been applied to materials in which the main components were respectively theophylline, digoxin, 17ß-estradiol, and aldrin. Its performance has been validated in international comparisons coordinated by the BIPM and is in principle applicable to a wide structural range of stable, nonvolatile organic compounds. It has been successfully applied to mass fraction assignments when the main component is present in the range of (950-1000) mg/g and can achieve associated standard uncertainties ranging from 0.5 mg/g (for high purity materials or those containing well-characterized, stable minor components) to 2 mg/g (materials with a significant number or variety of impurities). It is in principle equally applicable to materials with a smaller mass fraction content of the main component.

Temperature measurement and optical path-length bias improvement modifications to National Institute of Standards and Technology ozone reference standards.

UNLABELLED: Ambient ozone measurements in the United States and many other countries are traceable to a National Institute of Standards and Technology Standard Reference Photometer (NIST SRP). The NIST SRP serves as the highest level ozone reference standard in the United States, with NIST SRPs located at NIST and at many U.S. Environmental Protection Agency (EPA) laboratories. The International Bureau of Weights and Measures (BIPM) maintains a NIST SRP as the reference standard for international measurement comparability through the International Committee of Weights and Measures (CIPM). In total, there are currently NIST SRPs located in 20 countries for use as an ozone reference standard. A detailed examination of the NIST SRP by the BIPM and NIST has revealed a temperature gradient and optical path-length bias inherent in all NIST SRPs. A temperature gradient along the absorption cells causes incorrect temperature measurements by as much as 2 degrees C. Additionally, the temperature probe used for temperature measurements was found to inaccurately measure the temperature of the sample gas due to a self-heating effect. Multiple internal reflections within the absorption cells produce an actual path length longer than the measured fixed length used in the calculations for ozone mole fractions. Reflections from optical filters located at the exit of the absorption cells add to this effect. Because all NIST SRPs are essentially identical, the temperature and path-length biases exist on all units by varying amounts dependent upon instrument settings, laboratory conditions, and absorption cell window alignment. This paper will discuss the cause of and physical modifications for reducing these measurement biases in NIST SRPs. Results from actual NIST SRP bias upgrades quantifying the effects of these measurement biases on ozone measurements are summarized. IMPLICATIONS: NIST SRPs are maintained in laboratories around the world underpinning ozone measurement calibration and traceability within and between countries. The work described in this paper quantifies and shows the reduction of instrument biases in NIST SRPs improving their overall agreement. This improved agreement in all NIST SRPs provides a more stable baseline for ozone measurements worldwide.

Second harmonic generation on crystalline organic nanoclusters under extreme nanoconfinement in functionalized silica-benzil composites.

We demonstrate a series of organic-inorganic nanocomposite materials combining the mesoporous silica (PS) and benzil (BZL) nanocrystals embedded into its nanochannels (6.0-13.0 nm in diameter) by capillary crystallization. One aims to design novel, efficient nonlinear optical composite materials in which inactive amorphous host PS-matrix provides a tubular scaffold structure, whereas nonlinear optical functionality results from specific properties of the deposited guest BZL-nanocrystals. A considerable contraction of the BZL melt during its crystallization inside the silica nanochannels results in a formation of the texture consisting of (221)- and (003)-oriented BZL nanoclusters (22 nm in length), separated by voids. Specificity of the textural morphology similarly to the spatial confinement significantly influences the nonlinear optical features of composite PS:BZL materials being explored in the second harmonic generation (SHG) experiment. The light polarization anisotropy of the SHG response appears to be considerably reduced at channel diameters larger than 7 nm apparently due to the multiple scattering and depolarization of the light on randomly distributed and crystallographically oriented BZL-nanoclusters. The normalized SHG response decreases nonlinearly by more than one order of magnitude as the channel diameter decreases from 13.0 to 6.0 nm and vanishes when spatial cylindrical confinement approaches the sizes of a few molecular layers suggesting that the embedded BZL clusters indeed are not uniformly crystalline but are characterized by more complex morphology consisting of a disordered SHG-inactive amorphous shell, covering the channel wall, and SHG-active crystalline core. Understanding and controlling of the textural morphology in inorganic-organic nanocrystalline composites as well as its relationships with nonlinear optical properties can lead to the development of novel efficient nonlinear optical materials for the light energy conversion with prospective optoelectronic and photonic applications.

Highly accurate nitrogen dioxide (NO2) in nitrogen standards based on permeation.

The development and operation of a highly accurate primary gas facility for the dynamic production of mixtures of nitrogen dioxide (NO(2)) in nitrogen (N(2)) based on continuous weighing of a permeation tube and accurate impurity quantification and correction of the gas mixtures using Fourier transform infrared spectroscopy (FT-IR) is described. NO(2) gas mixtures in the range of 5 µmol mol(-1) to 15 µmol mol(-1) with a standard relative uncertainty of 0.4% can be produced with this facility. To achieve an uncertainty at this level, significant efforts were made to reduce, identify and quantify potential impurities present in the gas mixtures, such as nitric acid (HNO(3)). A complete uncertainty budget, based on the analysis of the performance of the facility, including the use of a FT-IR spectrometer and a nondispersive UV analyzer as analytical techniques, is presented in this work. The mixtures produced by this facility were validated and then selected to provide reference values for an international comparison of the Consultative Committee for Amount of Substance (CCQM), number CCQM-K74, (1) which was designed to evaluate the consistency of primary NO(2) gas standards from 17 National Metrology Institutes.

An SI-traceable reference material for virus-like particles.

A reference material for virus-like particles traceable to the International System of Units (Système International d'Unités - the SI) is reported. The material addresses the need for developing reference standards to benchmark virus-like gene delivery systems and help harmonize measurement approaches for characterization and testing. The material is a major component of synthetic polypeptide virus-like particles produced by the state-of-the-art synthetic and analytical chemistry methods used to generate gene delivery systems. The purity profile of the material is evaluated to the highest metrological order demonstrating traceability to the SI. The material adds to the emerging toolkit of reference standards for quantitative biology.

Correlation between Nonlinear Optical Effects and Structural Features of Aurone-Based Methacrylic Polymeric Thin Films.

In this study, new photonics architectures and aurone-based methacrylic polymers were designed and synthesized for their optical and nonlinear optical properties. The studied polymeric thin films were deposited by spin coating method. SHG and THG effects were measured via Maker fringe technique in transmission mode and determined using theoretical models. Investigations involved the theoretical quantum chemical calculation of dipole moments, frontier molecular orbital HOMO and LUMO energies, and first (ß) and second (γ) hyperpolarizabilities. We determined the impact of the substitution in the para position of the phenyl ring and at the dipole moment of the chromophore on the nonlinear optical properties of the investigated polymers. The presented theoretical and experimental studies provide important information with respect to the design of methacrylic-based polymeric thin film devices and supplement existing knowledge with respect to their nonlinear behaviour.