Candidate Reference Material Research Articles

Multiplatform metabolomic interlaboratory study of a whole human stool candidate reference material from omnivore and vegan donors.

Human metabolomics has made significant strides in understanding metabolic changes and their implications for human health, with promising applications in diagnostics and treatment, particularly regarding the gut microbiome. However, progress is hampered by issues with data comparability and reproducibility across studies, limiting the translation of these discoveries into practical applications. This study aims to evaluate the fit-for-purpose of a suite of human stool samples as potential candidate reference materials (RMs) and assess the state of the field regarding harmonizing gut metabolomics measurements. An interlaboratory study was conducted with 18 participating institutions. The study allowed for the use of preferred analytical techniques, including liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR). Different laboratories used various methods and analytical platforms to identify the metabolites present in human stool RM samples. The study found a 40% to 70% recurrence in the reported top 20 most abundant metabolites across the four materials. In the full annotation list, the percentage of metabolites reported multiple times after nomenclature standardization was 36% (LC-MS), 58% (GC-MS) and 76% (NMR). Out of 9,300 unique metabolites, only 37 were reported across all three measurement techniques. This collaborative exercise emphasized the broad chemical survey possible with multi-technique approaches. Community engagement is essential for the evaluation and characterization of common materials designed to facilitate comparability and ensure data quality underscoring the value of determining current practices, challenges, and progress of a field through interlaboratory studies.

Purity determination and uncertainty evaluation of L-selenium-methylselenocysteine

In our country, sodium selenite and sodium selenate are still used as nutritional fortifiers of selenium. Both of them are inorganic selenium, the absorption effect of human body is lower, the risk of toxic wind is larger, and it is easy to cause selenium poisoning. L-selenium-methylselenocysteine (L-SeMC) is a good source of organic selenium, which has higher biological activity and availability. At present, L-SeMC solid standard materials have not yet been developed, a high accuracy and low technical requirement method is needed to determine the purity of candidate reference materials (RMs). In this way, the key steps in the development of reference materials can be completed, and provide a source of high quality selenium supplement for people. In this paper, the candidate structures were identified and analyzed by high performance liquid chromatography (HPLC), Fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR). Homogeneity, together with stability were also measured by HPLC. The sample can be transported for a short period of one month under normal conditions, which can also be stored for a long time within the temperature range of −20 °C to room temperature for more than one year. The purity of L-SeMC measured by mass balance method and quantitative nuclear magnetic resonance (qNMR) was 99.5 % with an expanded uncertainty of 0.4 % (k = 2). However, differential scanning calorimetry (DSC) was not suitable for the purity determination of L-SeMC samples. This method has a wide range of applications and may provide a reference for the purity determination of other RMs.

Isolation of ovatoxin-a from Ostreopsis cf. ovata cultures. A key step for hazard characterization and risk management of ovatoxins

Ostreopsis cf. ovata, a benthic/epiphytic marine dinoflagellate, is currently spreading in tropical, sub-tropical and temperate areas, causing periodic Harmful Algal Blooms (HABs). It produces a wide array of palytoxin-like compounds named ovatoxins (OVTXs), with OVTX-a generally the most abundant congener. Despite numerous cases of human poisonings and environmental damage associated with the presence of OVTXs and O. cf. ovata proliferations, a complete characterization of the toxicity of this class of molecules cannot be performed until Reference Material (RM) for individual congeners is available. This, in turn, requires the availability of sufficient amounts of toxin at a high purity grade. To achieve this goal, herein an analytical re-evaluation of critical-steps of OVTX-a isolation from O. cf. ovata cell pellets is reported. The overall procedure consists of four steps, namely an extraction, a Medium Pressure Liquid Chromatography (MPLC) separation, and two preparative High Performance Liquid Chromatography (HPLC) steps. Particular attention was paid to the extraction step to evaluate the repeatability in OVTX-a yields. For subsequent steps, loading sample preparation and chromatographic conditions were refined. As a result, a significant increase in recovery yields (from 12.5 to 20 ± 3%) and in purity grade (from 51% to 94%) of the isolated OVTX-a was achieved in comparison to previous studies. The improved procedure can easily be applied to isolate sufficient quantities of a good candidate RM for OVTX-a.

Establishment of a Reference Material in Quality Control for Use in Infectivity and Identity Assays of Recombinant COVID-19 Vaccine, in Accordance with International Standards Organization Guidance.

The COVID-19 pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), began in 2019. One of the strategies for pandemic control was mass vaccination. In Brazil, the recombinant COVID-19 vaccine (RCV) was produced on a large scale and offered at no charge to the population. The specifications for quality control analyses of RCV included identity and infectivity determination. To validate the results, a reference material (RM) must be analyzed in parallel with the sample vaccine. This research aimed to establish the RM for use in the identity and infectivity assay for RCV. The candidate RM was analyzed using homogeneity and stability studies. The RM was considered homogeneous for identity (cycle threshold (Ct) ≤ 25.19) and infectivity (average x- was 9.25 log10 infectious units/mL). The RM was considered adequately stable for identity during the total period in all studies, being stable at -70, 5, and 22.5 °C for 380, 313, and 14 days, respectively (Ct ≤ 21.81). For infectivity, the RM was stable at -70, 5, and 22.5 °C for 380, 97, and three days, respectively. Since the property identity and infectivity values of the RM were established, the new RM could be used in quality control analysis.

The Alzheimer's Association Global Biomarker Standardization Consortium (GBSC) plasma phospho-tau Round Robin study.

Phosphorylated tau (p-tau) is a specific blood biomarker for Alzheimer's disease (AD) pathology. Multiple p-tau biomarkers on several analytical platforms are poised for clinical use. The Alzheimer's Association Global Biomarker Standardisation Consortium plasma phospho-tau Round Robin study engaged assay developers in a blinded case-control study on plasma p-tau, aiming to learn which assays provide the largest fold-changes in AD compared to non-AD, have the strongest relationship between plasma and cerebrospinal fluid (CSF), and show the most consistent relationships between methods (commutability) in measuring both patient samples and candidate reference materials (CRM). Thirty-three different p-tau biomarker assays, built on eight different analytical platforms, were used to quantify paired plasma and CSF samples from 40 participants. AD biomarker status was categorised as "AD pathology" (n=25) and "non-AD pathology" (n=15) by CSF Aβ42/Aβ40 (US-FDA; CE-IVDR) and p-tau181 (CE-IVDR) methods. The commutability of four CRM, at three concentrations, was assessed across assays. Plasma p-tau217 consistently demonstrated higher fold-changes between AD and non-AD pathology groups, compared to other p-tau epitopes. Fujirebio LUMIPULSE G, UGOT IPMS, and Lilly MSD p-tau217 assays provided the highest median fold-changes. In CSF, p-tau217 assays also performed best, and exhibited substantially larger fold-changes than their plasma counterparts, despite similar diagnostic performance. P-tau217 showed the strongest correlations between plasma assays (rho=0.81 to 0.97). Plasma p-tau levels were weakly-to-moderately correlated with CSF p-tau, and correlations were non-significant within the AD group alone. The evaluated CRM were not commutable across assays. Plasma p-tau217 measures had larger fold-changes and discriminative accuracies for detecting AD pathology, and better agreement across platforms than other plasma p-tau variants. Plasma and CSF markers of p-tau, measured by immunoassays, are not substantially correlated, questioning the interchangeability of their continuous relationship. Further work is warranted to understand the pathophysiology underlying this dissociation, and to develop suitable reference materials facilitating cross-assay standardisation. Alzheimer's Association (#ADSF-24-1284328-C).

Identification by HSQC and quantification by qHNMR innovate pharmaceutical amino acid analysis

This study introduces a new NMR-based methodology for identification (ID) and quantification (purity, strength) assays of widely used amino acids. A detailed analysis of four amino acids and their available salts was performed with both a high-field (600 MHz) and a benchtop (60 MHz) NMR instrument. To assess sensitivity constraints, samples for 1H NMR analysis were initially prepared using only 10 mg of analyte and 1 mg of maleic acid (MA) as an internal calibrant (IC) and secondary chemical shift reference. The characteristic dispersion of the peak patterns indicating the presence or absence of a counterion (mostly chloride) was conserved at both high and low-field strength instruments, showing that the underlying NMR spectroscopic parameters, i.e., chemical shifts and coupling constants, are independent of the magnetic field strength. However, as the verbal descriptions of 1H NMR spectra are challenging in the context of reference materials and pharmaceutical monographs, an alternative method for the identification (ID) of amino acids is proposed that uses 13C NMR patterns from multiplicity-edited HSQC (ed-HSQC), which are both compound-specific and straightforward to document. For ed-HSQC measurements, the sample amount was increased to 30 mg of the analyte and several acquisition parameters were tested, including t1 increments used in the pulse program, number of scans, and repetition time. Excellent congruence with deviations <0.1 ppm was achieved for the 13C chemical shifts from 1D 13C NMR spectra (150 MHz) vs. those extracted from ed-HSQC (15 MHz traces). Finally, all samples of amino acid candidate reference materials were quantified by 1H qNMR (abs-qHNMR) at both 600 and 60 MHz. At high field, both IC and relative quantitations were performed, however, with the low-field instrument, only the IC method was used. The results showed that the analyzed reference material candidates were generally highly pure compounds. To achieve adequately low levels of uncertainty for such high-purity materials, the sample amounts were increased to 100 mg of analytes and 10 mg of the IC and replicates were analyzed for selected amino acids.

Evaluating the Commutability of Reference Materials for α-Fetoprotein: Accurate Value Assignment With Multiple Systems and Trueness Verification.

The accurate measurement of α-fetoprotein (AFP) is critical for clinical diagnosis. However, different AFP immunoassays may yield different results. Appropriate AFP reference materials (RMs) were selected and assigned accurate values for applications with external quality assessment (EQA) programs to standardize AFP measurements. Forty individual clinical samples and six different concentrations of candidate RMs (Can-RMs, L1-L6) were prepared by the Beijing Center for Clinical Laboratories. The Can-RMs were assigned target values by performing five immunoassays, using WHO International Standard 72/225 as a calibrator, and sent to 45 clinical laboratories in Beijing for AFP measurements. The commutability of all RMs was assessed based on CLSI and the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) approaches. Analytical performance was assessed for compliance based on accuracy (total error, TE), trueness (bias), and precision (CV). The Can-RMs were commutable for all immunoassays using the CLSI approach and for 6 of 10 assay combinations using the IFCC approach. RMs diluted in WHO RM 72/225 were commutable among all assays with the CLSI approach, except for serum matrix (Autolumo vs. Roche analyzer) and diluted water matrix (Abbott vs. Roche/Mindray analyzer), whereas some inconclusive and non-commutable results were found using the IFCC approach. The average pass rates based on the TE, bias, and CV were 91%, 81%, and 95%, respectively. The commutability of the RMs differed between both evaluation approaches. The Can-RMs exhibited good commutability with the CLSI approach, suggesting their suitability for use with that approach as commutable EQA materials with assigned values and for monitoring the performance of AFP measurements.

A Comparative Study of Analytical Methods for Medium-Chain Chlorinated Paraffins Using a Candidate Reference Material

A Comparative Study of Analytical Methods for Medium-Chain Chlorinated Paraffins Using a Candidate Reference Material

Towards a reference material for microplastics’ number concentration—case study of PET in water using Raman microspectroscopy

Increasing demand for size-resolved identification and quantification of microplastic particles in drinking water and environmental samples requires the adequate validation of methods and techniques that can be used for this purpose. In turn, the feasibility of such validation depends on the existence of suitable certified reference materials (CRM). A new candidate reference material (RM), consisting of polyethylene terephthalate (PET) particles and a water matrix, has been developed. Here, we examine its suitability with respect to a homogeneous and stable microplastic particle number concentration across its individual units. A measurement series employing tailor-made software for automated counting and analysis of particles (TUM-ParticleTyper 2) coupled with Raman microspectroscopy showed evidence of the candidate RM homogeneity with a relative standard deviation of 12% of PET particle counts involving particle sizes >30 µm. Both the total particle count and the respective sums within distinct size classes were comparable in all selected candidate RM units. We demonstrate the feasibility of production of a reference material that is sufficiently homogeneous and stable with respect to the particle number concentration.Graphical

Certification of New Selenium-Enriched Yeast and Supplement Reference Materials for Selenomethionine Using Two Independent Measurement Strategies.

Selenium-enriched yeast possesses the unique ability of transforming chemical selenium, such as sodium selenite, into a biologically active form, which mitigates its toxic effects on the human body. The transformation product of this process, selenomethionine, can be safely and effectively absorbed and utilized by the human body; hence, it has been spiked into a selenium-enriched supplement. This study employs two distinct measurement strategies to determine the selenomethionine content in two candidate reference materials, a selenium-enriched yeast powder and supplement, using both organic and inorganic mass spectrometry. The concentrations of selenomethionine in the selenium-enriched yeast were determined using HPLC-ICP-MS and HPLC- ESI-MS/MS, with mass fractions measured at 718 mg SeMet kg-1 and 715 mg SeMet kg-1, respectively. Notably, both methods yielded consistent results for the selenium supplement, with a selenomethionine mass fraction of 59 mg SeMet kg-1. Ultimately, the certified values of these candidate reference materials were determined as 716 mg kg-1 and 59 mg SeMet kg-1 with expanded uncertainties of 36 mg SeMet kg-1 (k = 2) and 5 mg SeMet kg-1 (k = 2), respectively. The development of these candidate reference materials serves as a valuable reference for diverse methods aiming to determine the value of organic selenium speciation in complex food substrates.

Morphologically-Directed Raman Spectroscopy as an Analytical Method for Subvisible Particle Characterization in Therapeutic Protein Product Quality

Subvisible particles (SVPs) are a critical quality attribute of injectable therapeutic proteins (TPs) that needs to be controlled due to potential risks associated with drug product quality. The current compendial methods routinely used to analyze SVPs for lot release provide information on particle size and count. However, chemical identification of individual particles is also important to address root-cause analysis. Herein, we introduce Morphologically-Directed Raman Spectroscopy (MDRS) for SVP characterization of TPs. The following particles were used for method development: (1) polystyrene microspheres, a traditional standard used in industry; (2) photolithographic (SU-8); and (3) ethylene tetrafluoroethylene (ETFE) particles, candidate reference materials developed by NIST. In our study, MDRS rendered high-resolution images for the ETFE particles (> 90%) ranging from 19 to 100 μm in size, covering most of SVP range, and generated comparable morphology data to flow imaging microscopy. Our method was applied to characterize particles formed in stressed TPs and was able to chemically identify individual particles using Raman spectroscopy. MDRS was able to compare morphology and transparency properties of proteinaceous particles with reference materials. The data suggests MDRS may complement the current TPs SVP analysis system and product quality characterization workflow throughout development and commercial lifecycle.

Commutability assessment of candidate reference materials for plasma renin activity measurement: current challenges.

This study aims to evaluate the commutability of external quality assessment (EQA) materials and candidate reference materials (RMs) for plasma renin activity (PRA) assay. Commutabilities of 16 candidate RMs were measured along with 40 clinical samples by the four different routine PRA assays, including three LC‒MS/MS assays and onechemiluminescence immunoassay. Sixteen candidate RMs included native/spiked human plasma pools (small-scale pools with <50 individuals) and current EQA materials (large-scale pools with >1,000 individuals). Difference inbias approach and linear regression with prediction interval approach were adopted to determine the commutability. Two-way variance analysis was used to estimate the effects of spiked and pool size on the commutability. Stability and homogeneity studies were performed. Precision and correlation performance of all assays was acceptable. In the difference in bias approach, thecommutability results were not satisfactory (noncommutability: 14/16) and significant sample-specific effects were detected in assay pairs using different incubation buffers. For the prediction interval approach, no commutability was observed in the spiked small-scale pools; EQA materials (4/9) had more satisfactory commutability among all assays than the small-scale pools (2/7); RMs of large-scale pools tend to have better commutability no matter spiked ornot. Commutable RMs were obtainable but challenging. Current EQA materials with relatively good commutability, stability, and homogeneity were appropriate RMs. Large-scale pools are tending to be commutable. Spiking in small-scale pools was not suggested to prepare RMs. MPs adopting a uniform incubation buffer would be preferable for further commutability research.

Commutability evaluation of candidate reference materials and ERM-DA470k/IFCC for immunoglobulin M using two international approaches.

This study aimed to assess the commutability of frozen pooled human serum (PHS), high concentration of Immunoglobulin M (IgM) pure diluted materials (HPDM), commercialized pure materials (CPM), and dilutions of ERM-DA470k/IFCC in IgM detection using the CLSI and IFCC approaches, to support standardization or harmonization of IgM measurement. Twenty-four serum samples, relevant reference materials (PHS, HPDM, CPM), and different ERM-DA470k/IFCC dilutions were analyzed in triplicate using six routine methods. The commutability of the relevant reference materials was carried out following CLSI EP30-A and IFCC bias analysis. According to the CLSI approach, low, medium, and high concentrations of PHS, HPDM, and CPM were commutable on 10, 13, 15, 13, and 8 of 15 assay combinations, respectively. Using the IFCC approach, low, medium, and high concentrations of PHS, HPDM, and CPM were commutable on 10, 11, 9, 15, and 10 of 15 assay combinations, respectively. The ERM-DA470k/IFCC dilutions with D-PBS and RPMI-1640 Medium were commutable on 13 of 15 assay combinations according to CLSI and were commutable on all 15 assay combinations using IFCC approach. High concentration of PHS were commutable on all six detection systems using the CLSI approach. Low and medium concentration of PHS showed unsatisfied commutability. HPDM, not CPM have good commutability, has the potential to become reference materials. ERM-DA470k/IFCC diluted with different medium showed different commutability.

Comparison and commutability study among four faecal immunochemical tests (FIT) systems.

Faecal immunochemical tests for haemoglobin (FIT) are used in colorectal cancer screening programs around the world and increasingly for triage of symptomatic patients. FIT results are currently not traceable to a common reference standard and results obtained on various FIT systems may not be equivalent. The size of the bias between the systems is difficult to quantify due to the complex pre-analytical aspects of FIT. This study aimed to quantify the bias and the correlation between four FIT systems by measuring a panel of 38 faecal samples while limiting the effect of the pre-analytical aspects. In addition, the commutability of seven candidate reference materials (RM) was assessed. Pairwise method comparisons based on faecal samples demonstrated Pearson correlation coefficients ranging between 0.944 and 0.970 and an average proportional bias of-30 to-35 % for one FIT system compared to the other three. The relative standard deviation among biases of the individual samples was around 20 %. Due to these sample specific differences, no decisive conclusions could be drawn in the commutability study. However, twocandidate RMs, prepared in the FIT system-specific storage/extraction buffers, had a better commutable profile than the other five. The use of a common threshold for all FIT systems is currently not possible due to the presence of a proportional bias. We have identified potential commutable RMs to take to further studies on the production of a common calibrator, with the aim being to reduce the analytical bias observed on different FIT systems.

Accurate method for value assignment of carcinoembryonic antigen reference materials.

In this study, we explored the commutability of reference materials (RMs) for carcinoembryonic antigen (CEA), selected the appropriate diluent matrix of the first International Reference Preparation (IRP) 73/601 of the World Health Organization (WHO 73/601) for CEA, and improved the comparability of CEA measurement results among different assay systems. Forty serum samples were divided into five aliquots. WHO 73/601 was diluted into nine concentrations using five diluents with different components, and the candidate RMs for CEA at five concentrations (C1-C5) were prepared by the Beijing Clinical Laboratory Center (BCCL). The samples were analyzed via five automated CEA immunoassays. Carcinoembryonic antigen candidate RMs were commutable among all immunoassays based on the CLSI approach and among 7 of 10 assay combinations based on the IFCC approach. WHO 73/601 diluted in phosphate-buffered saline (PBS) was commutable among all assays based on the CLSI approach and among 5 of 10 pairwise comparisons based on the IFCC approach with correction of bias at diluted concentrations, except for the lowest concentration, which had the smallest variation among systems. The median percentage biases among assays were decreased after calibration. The BCCL candidate RMs (C2-C5) for CEA were commutable among all immunoassays. WHO 73/601 RMs diluted in a PBS buffer matrix were selected as common calibrators for five immunoassays, which reduced bias, thereby effectively improving the harmonization of CEA detection; therefore, they could be used to assign values to CEA candidate RMs developed by BCCL. Our findings promote the harmonization of CEA detection in immunoassays.

Establishment of certified reference material for the potency assay in yellow fever vaccine quality control, in accordance with International Standards Organization guidance

The attenuated yellow fever vaccine (YFV) is offered free of charge to the Brazilian population through the National Immunization Program (NIP). One of the specifications for quality control analyses of the vaccine is the potency determination. This test determines the number of plaque forming units (PFU) in Vero cells. In order to validate the results, the reference material (RM) is analysed in parallel with an established reference vaccine. The aim of this study was to establish certified RM for use as an internal control in the potency assay for the production chain of YFV. The candidate RM homogeneity and stability were determined, and characterized by a collaborative study for further certification. The RM was considered sufficiently homogeneous with average 4.68 log10 IU/HD and stable at (−20 ± 10) ºC and (22.5 ± 2.5) ºC for 715 and 183 days, respectively. When reconstituted and stored in aliquots of 0.6 mL, it was stable at (−20 ± 10) ºC for eight days. But it was not stable at (5 ± 3) ºC for three days. In a collaborative study, two independents’ laboratories gave an averaged value of 4.56 ± 0.030 log10 IU/HD. After determining the expanded uncertainty of homogeneity, stability, and characterization, the certified RM lot: 195VFA020Z presented a property value of 4.56 ± 0.22 log10 IU/HD. It was concluded that the new certified RM can be used in routine analysis of a YFV producer, since it has its property value established and it is stable. The possibility of using it in aliquots after reconstitution will also allow the RM to have a much longer shelf life.

Reference Materials for the Phase Transition Temperatures (Curie Temperature) Based on Alumel, Nickel and Iron Silicide

The requirements for thermal analysis instruments are increasing in terms of their metrological support with the development of scientific and technological progress in the field of metrological supervision and electronics industry. The study of determining the phase transition temperature, namely the Curie temperature, is of particular relevance for the metrological support of the applied thermal analysis instruments and traceability to the basic physical units.The purpose of the research was to test the possibility of using materials based on alumel, nickel and iron silicide (trafoperm) as phase transition temperature standards for candidate reference materials of phase transition temperatures (RMs) traceable to the SI unit of the «temperature» value.The procedure for measuring the phase transition temperatures (Curie temperature – TC) was carried out by the thermomagnetometric method using an STA 449 F5 JUPITER thermal analyzer from the State Primary Standard GET 173-2017. The determination of the RM certified value was carried out in accordance with GOST ISO Guide 35–2015, the contributions to the uncertainty from the heterogeneity of the initial materials were evaluated, and the short-term and long-term stability of the materials were studied.A comparison of the certified values of the developed RMs with the reference values of the Curie temperature showed that they are consistent.The theoretical significance of the results obtained is proof of the possibility of using the thermomagnetometric method for the development of certified reference materials of phase transition temperatures GSO 12005–2022/GSO 12007–2022. The possibility of applying the method of differential scanning calorimetry in determining the Curie temperature was also confirmed.The practical significance of the results obtained allows expanding the possibilities for constructing the calibration dependence of measuring instruments for thermogravimetric analysis and monitoring its stability, as well as increasing the measurement precision of the Curie temperature of various substances and materials.

Lipid Nanoparticle and Liposome Reference Materials: Assessment of Size Homogeneity and Long-Term -70 °C and 4 °C Storage Stability.

With recent advances and anticipated proliferation of lipid nanoparticle (LNP)-delivered vaccines and therapeutics, there is a need for the availability of internationally recognized reference materials of LNP systems. Accordingly, we developed six LNP and liposome (anionic, neutral, and cationic each) candidate reference material formulations and thoroughly characterized by dynamic light scattering their particle hydrodynamic size (Z-avr) and polydispersity. We also evaluated the particle size homogeneity and long-term -70 °C and 4 °C storage stability using multiple large sets of randomly selected vials for each formulation. The formulations stored at -70 °C remained stable and homogeneous for a minimum of 9 months. The Z-avr relative combined uncertainty and the long-term variability were both <1.3% for liposome formulations and anionic LNPs, (3.9% and 1.7%) for neutral LNPs, and (6.7% and 4.4%) for cationic LNPs. An inadvertent few-hour-long storage temperature increase to -35 °C due to a freezer malfunction resulted in a small change of the size and size distribution of anionic liposomes and LNPs but, unexpectedly, a larger size increase of the neutral and cationic liposomes (≤5%) and LNPs (≤25%). The mean Z-avr values of the LNPs stored at 4 °C appeared to slowly increase with t1/3, where t is the storage time, and the Z-avr between-vial heterogeneity and mean polydispersity index values appeared to decrease; no change was observed for liposomes. The size and size distribution evolution of LNPs stored at 4 °C was attributed to an incomplete equilibration of the formulations following the addition of sucrose prior to the initial freezing. Such a process of size increase and size distribution narrowing has not been previously discussed nor observed in the context of LNPs.

Assessing the commutability of candidate reference materials for the harmonization of neurofilament light measurements in blood.

Neurofilament light chain (NfL) concentration in blood is a biomarker of neuro-axonal injury in the nervous system and there now exist several assays with high enough sensitivity to measure NfL in serum and plasma. There is a need for harmonization with the goal of creating a certified reference material (CRM) for NfL and an early step in such an effort is to determine the best matrix for the CRM. This is done in a commutability study and here the results of the first one for NfL in blood is presented. Forty paired individual serum and plasma samples were analyzed for NfL on four different analytical platforms. Neat and differently spiked serum and plasma were evaluated for their suitability as a CRM using the difference in bias approach. The correlation between the different platforms with regards to measured NfL concentrations were very high (Spearman's ρ≥0.96). Samples spiked with cerebrospinal fluid (CSF) showed higher commutability compared to samples spiked with recombinant human NfL protein and serum seems to be a better choice than plasma as the matrix for a CRM. The results from this first commutability study on NfL in serum/plasma showed that it is feasible to create a CRM for NfL in blood and that spiking should be done using CSF rather than with recombinant human NfL protein.

Commutability Assessment of Candidate Reference Materials for Lipoprotein(a) by Comparison of a MS-based Candidate Reference Measurement Procedure with Immunoassays.

Elevated concentrations of lipoprotein(a) [Lp(a)] are directly related to an increased risk of cardiovascular diseases, making it a relevant biomarker for clinical risk assessment. However, the lack of global standardization of current Lp(a) measurement procedures (MPs) leads to inconsistent patient care. The International Federation for Clinical Chemistry and Laboratory Medicine working group on quantitating apolipoproteins by mass spectrometry (MS) aims to develop a next-generation SI (International system of units)-traceable reference measurement system consisting of a MS-based, peptide-calibrated reference measurement procedure (RMP) and secondary serum-based reference materials (RMs) certified for their apolipoprotein(a) [apo(a)] content. To reach measurement standardization through this new measurement system, 2 essential requirements need to be fulfilled: a sufficient correlation among the MPs and appropriate commutability of future serum-based RMs. The correlation among the candidate RMP (cRMP) and immunoassay-based MPs was assessed by measuring a panel of 39 clinical samples (CS). In addition, the commutability of 14 different candidate RMs was investigated. Results of the immunoassay-based MPs and the cRMPs demonstrated good linear correlations for the CS but some significant sample-specific differences were also observed. The results of the commutability study show that RMs based on unspiked human serum pools can be commutable with CS, whereas human pools spiked with recombinant apo(a) show different behavior compared to CS. The results of this study show that unspiked human serum pools are the preferred candidate secondary RMs in the future SI-traceable Lp(a) Reference Measurement System.