Interlaboratory Comparison Study Research Articles

Interlaboratory Comparison of Antibody-Free LC-MS/MS Measurements of C-peptide and Insulin.

The enhanced precision and selectivity of liquid chromatography-tandem mass spectrometry (LC-MS/MS) makes it an attractive alternative to certain clinical immunoassays. Easily transferrable work flows could help facilitate harmonization and ensure high-quality patient care. We aimed to evaluate the interlaboratory comparability of antibody-free multiplexed insulin and C-peptide LC-MS/MS measurements. The laboratories that comprise the Targeted Mass Spectrometry Assays for Diabetes and Obesity Research (TaMADOR) consortium verified the performance of a validated peptide-based assay (reproducibility, linearity, and lower limit of the measuring interval [LLMI]). An interlaboratory comparison study was then performed using shared calibrators, de-identified leftover laboratory samples, and reference materials. During verification, the measurements were precise (2.7% to 3.7%CV), linear (4 to 15 ng/mL for C-peptide and 2 to 14 ng/mL for insulin), and sensitive (LLMI of 0.04 to 0.10 ng/mL for C-peptide and 0.03 ng/mL for insulin). Median imprecision across the 3 laboratories was 13.4% (inter-quartile range [IQR] 11.6%) for C-peptide and 22.2% (IQR 20.9%) for insulin using individual measurements, and 10.8% (IQR 8.7%) and 15.3% (IQR 14.9%) for C-peptide and insulin, respectively, when replicate measurements were averaged. Method comparison with the University of Missouri reference method for C-peptide demonstrated a robust linear correlation with a slope of 1.044 and r2 = 0.99. Our results suggest that combined LC-MS/MS measurements of C-peptide and insulin are robust and adaptable and that standardization with a reference measurement procedure could allow accurate and precise measurements across sites, which could be important to diabetes research and help patient care in the future.

PUM1 and PGK1 are Favorable Housekeeping Genes over Established Biodosimetry-related Housekeeping Genes such as HPRT1, ITFG1, DPM1, MRPS5, 18S rRNA and Others after Radiation Exposure.

In gene expression (GE) studies, housekeeping genes (HKGs) are required for normalization purposes. In large-scale inter-laboratory comparison studies, significant differences in dose estimates are reported and divergent HKGs are employed by the teams. Among them, the 18S rRNA HKG is known for its robustness. However, the high abundance of 18S rRNA copy numbers requires dilution, which is time-consuming and a possible source of errors. This study was conducted to identify the most promising HKGs showing the least radiation-induced GE variance after radiation exposure. In the screening stage of this study, 35 HKGs were analyzed. This included selected HKGs (ITFG1, MRPS5, and DPM1) used in large-scale biodosimetry studies which were not covered on an additionally employed pre-designed 96-well platform comprising another 32 HKGs used for different exposures. Altogether 41 samples were examined, including 27 ex vivo X-ray irradiated blood samples (0, 0.5, 4 Gy), six X-irradiated samples (0, 0.5, 5 Gy) from two cell lines (U118, A549), as well as eight non-irradiated tissue samples to encompass multiple biological entities. In the independent validation stage, the most suitable candidate genes were examined from another 257 blood samples, taking advantage of already stored material originating from three studies. These comprise 100 blood samples from ex vivo X-ray irradiated (0-4 Gy) healthy donors, 68 blood samples from 5.8 Gy irradiated (cobalt-60) Rhesus macaques (RM) (LD29/60) collected 0-60 days postirradiation, and 89 blood samples from chemotherapy-(CTx) treated breast tumor patients. CTx and radiation-induced GE changes in previous studies appeared comparable. RNA was isolated, converted into cDNA, and GE was quantified employing TaqMan assays and quantitative RT-PCR. We calculated the standard deviation (SD) and the interquartile range (IQR) as measures of GE variance using raw cycle threshold (Ct) values and ranked the HKGs accordingly. Dose, time, age, and sex-dependent GE changes were examined employing the parametrical t-test and non-parametrical Kruskal Wallis test, as well as linear regression analysis. Generally, similar ranking results evolved using either SD or IQR GE measures of variance, indicating a tight distribution of GE values. PUM1 and PGK1 showed the lowest variance among the first ten most suitable genes in the screening phase. MRPL19 revealed low variance among the first ten most suitable genes in the screening phase only for blood and cells, but certain comparisons indicated a weak association of MRPL19 with dose (P = 0.02-0.09). In the validation phase, these results could be confirmed. Here, IQR Ct values from, e.g., X-irradiated blood samples were 0.6 raw Ct values for PUM1 and PGK1, which is considered to represent GE differences as expected due to methodological variance. Overall, when compared, the GE variance of both genes was either comparable or lower compared to 18S rRNA. Compared with the IQR GE values of PUM1 and PGKI, two-fivefold increased values were calculated for the biodosimetry HKG HPRT1, and comparable values were calculated for biodosimetry HKGs ITFG1, MRPS5, and DPM1. Significant dose-dependent associations were found for ITFG1 and MRPS5 (P = 0.001-0.07) and widely absent or weak (P = 0.02-0.07) for HPRT1 and DPM1. In summary, PUM1 and PGK1 appeared most promising for radiation exposure studies among the 35 HKGs examined, considering GE variance and adverse associations of GE with dose.

Development of an Ephedrine In-House Matrix Reference Material and Its Application to Doping Analysis.

Biomatrix-based reference materials (RMs) improve the quality of laboratory test results by better representing actual samples. However, a matrix RM of ephedrine (EP) for threshold substances that require accurate analysis results has not yet been developed. Therefore, this study aimed to develop an in-house matrix RM for EP and subsequently apply it to analytical procedures. During the development of the in-house matrix EP RM, the system underwent homogeneity and stability studies. Additionally, it was subjected to interlaboratory comparison study in 11 laboratories, including 10 World Anti-Doping Agency (WADA)-accredited laboratories and our laboratory. Stability testing revealed no significant changes in the RM characteristics. For homogeneity, 10 random batches out of 200 were analyzed to confirm the uniformity within and between bottles. These results, combined with data from 11 laboratories, ensured retroactive validation. The traceability value of the in-house matrix EP RM was assigned as 9.83 ± 0.57 μg/mL (k = 2) by interlaboratory comparison studies and traceable uncertain evaluation. The feasibility of this method as a single calibration standard was confirmed in two laboratories. This substance is reliable and consistent for quality control during EP quantification, ensuring accurate and trustworthy outcomes. Consequently, this study establishes a framework and guidelines for producing in-house matrix RMs and serves as a reference for generating similar matrix RMs in other contexts.

Quantification of SARS-CoV-2 monoclonal IgG mass fraction by isotope dilution mass spectrometry.

The availability of serology assays to measure antibodies against the SARS coronavirus 2 (SARS-CoV-2) expanded rapidly during the Covid-19 pandemic. The interchangeable use of such assays to monitor disease progression and immune protection requires their standardization, for which suitably characterized monoclonal antibody materials can be useful. The methods, based on isotope dilution mass spectrometry, to value assign the mass fraction of such a material in solution within the context of an international interlaboratory comparison study (CCQM-P216) are described. The mass fraction in solution of a humanized IgG monoclonal antibody (mAb) against the SARS-CoV-2 Spike glycoprotein in the study sample has been value assigned through a combination of liquid chromatography, isotope dilution mass spectrometry (LC-ID-MS) methods and size exclusion chromatography with UV detection (SEC-UV). The former were developed for the quantification of amino acids and proteotypic peptides as surrogate analytes of the mAb while the latter was applied for the determination of the relative monomeric mass fraction. High-resolution mass spectrometry (hrMS) allowed the molecular weight evaluation and ruled out the presence of significant impurities. Method trueness was assessed using a subclass homologous IgG1 material value assigned by amino acid analysis. The assigned mass fraction of monomeric SARS-CoV-2 IgG in solution was 390 ± 16mg/g. The associated expanded uncertainty originated mainly from acid hydrolysis variability and Trypsin/Lys-C digestion variability and efficiency.

Evaluation of five multisteroid LC‒MS/MS methods used for routine clinical analysis: comparable performance was obtained for nine analytes.

A mass spectrometry (LC‒MS/MS)-based interlaboratory comparison study was performed for nine steroid analytes with five participating laboratories. The sample set contained 40 pooled samples of human serum generated from preanalyzed leftovers. To obtain a well-balanced distribution across reference intervals of each steroid, the leftovers first underwent a targeted mixing step. All participants measured a sample set once using their own multianalyte protocols and calibrators. Four participants used in-house developed measurement platforms, including IVD-CE certified calibrators, which were used by three participants; the 5th lab used the whole LC‒MS kit from an IVD manufacturer. All labs reported results for 17-hydroxyprogesterone, androstenedione, cortisol, and testosterone, and four labs reported results for 11-deoxycortisol, corticosterone, cortisone, dehydroepiandrosterone sulfate (DHEAS), and progesterone. Good or acceptable overall comparability was found in Bland‒Altman and Passing‒Bablok analyses. Mean bias against the overall mean remained less than ±10 % except for DHEAS, androstenedione, and progesterone at one site and for cortisol and corticosterone at two sites (max.-18.9 % for androstenedione). The main analytical problems unraveled by this study included a bias not previously identified in proficiency testing, operator errors, non-supported matrix types and higher inaccuracy and imprecision at lower ends of measuring intervals. This study shows that intermethod comparison is essential for monitoring the validity of an assay and should serve as an example of how external quality assessment could work in addition to organized proficiency testing schemes.

Postirradiation temperature influences DSB repair and dicentric chromosome formation-potential impact for dicentric chromosome analysis in interlaboratory comparisons.

The objective was to investigate the influence of different pre-storage temperatures in the dicentric chromosome analysis (DCA) protocol (22°C vs. 37°C) by using γ-H2AX+53BP1 foci as a marker for deoxyribonucleic acid (DNA) double-strand break (DSB) damage induction and repair and the formation of dicentric chromosomes as a result of mis-repair. Repair of γ-H2AX+53BP1 DSB foci was absent in samples that were incubated for 2h at 22°C after exposure of 0.5 and 1.2Gy. When 0.5- and 1.2-Gy-exposed samples were incubated at 37°C for 2h, there was an average decline of 31 and 52% of DSB foci, respectively. This indicated that DNA repair occurred. There was a 27% decrease in dicentric chromosome yield at 1.2Gy and a 15% decrease at 3.5Gy after post-irradiation incubation for 2 h at 37°C relative to the observed dicentric frequencies at 22°C. Recommended to re-phase: our data suggested that there were more open DSBs after a 2-h incubation at 22°C, which contributed to more mis-repair and dicentric formation from the start of culture. Our findings are corroborated by publications showing that lesion interaction based on enzymatic activity is suppressed below 21°C. As such temperature variations can be a source of variation in DCA during interlaboratory comparison studies, we propose to establish a common guide for the standardisation of pre-culture conditions in cytogenetic dosimetry proficiency testing.

Development of a certified reference material for accurate determination of the leaching of Pb and Zn in solid waste.

Certified reference materials (CRMs) with high accuracy and traceability play a significant role in the calibration of equipment and validation of analytical methods. However, there is still a lack of suitable solid waste CRMs for quality assurance and quality control. Thus, a CRM (GBW(E)085538) was developed for accurate determination and reliable measurement of the leaching of Pb and Zn in solid waste according to the requirements of ISO 17034 and the recommendations of ISO Guide 35. This study describes the steps performed for the development of the CRM. These steps include material preparation, homogeneity, and stability during transport and storage, assignment of certified values, and their uncertainties. The material was dried, ground, sieved and well-mixed, and the final bulk material was bottled in 1kg portions. Analytical techniques like inductively coupled plasma-mass spectrometry (ICP-MS), inductively coupled plasma-optical emission spectrometry (ICP-OES), and flame atomic absorption spectrometry (AAS) have been used for the characterization of property values. Concurrently, an inter-laboratory comparison study involving 9 qualified laboratories was implemented to support the certification study. The certified values of Pb and Zn were (4.66 ± 0.21) mg/L and (2.95 ± 0.14) mg/L with 7-month stability.

Standardizing In Vitro β-Lactam Antibiotic Allergy Testing with Synthetic IgE.

The global prevalence of β-lactam allergy poses a major challenge in administering first-line antibiotics, such as penicillins, to a significant portion of the population. The lack of β-lactam IgE antibody pools with defined selectivity hampers the standardization and validation of in vitro assays for β-lactam allergy testing. To address this limitation, this study introduces a synthetic IgE specific to β-lactam antibiotics as a valuable tool for drug allergy research and diagnostic tests. Using phage display technology, we constructed a library of human single-chain antibody fragments (scFv) to target the primary determinant of amoxicillin, a widely used β-lactam antibiotic. Subsequently, we produced a complete human synthetic IgE molecule using the highly efficient baculovirus expression vector system. This synthetic IgE molecule served as a standard in an in vitro chemiluminescence immunoassay for β-lactam antibiotic allergy testing. Our results demonstrated a detection limit of 0.05 IU/mL (0.63 pM), excellent specificity (100%), and a four-fold higher clinical sensitivity (73%) compared to the in vitro reference assay when testing a cohort of 150 serum samples. These findings have significant implications for reliable interlaboratory comparison studies, accurate labeling of allergic patients, and combating the global public health threat of antimicrobial resistance. Furthermore, by serving as a valuable trueness control material, the synthetic IgE facilitates the standardization of diagnostic tests for β-lactam allergy and demonstrates the potential of utilizing this synthetic strategy as a promising approach for generating reference materials in drug allergy research and diagnostics.

Development of a novel certified reference material for the determination of polycyclic aromatic hydrocarbons (PAHs) in whey protein powder

Matrix-based certified reference materials (CRMs) play a central role in the analysis of food contaminants for ensuring measurement accuracy and comparability, as they exhibit the same matrix effects during sample preparation and measurement as the food sample under investigation. However, the availability of such CRMs is still limited. This is also true for matrix CRMs containing polycyclic aromatic hydrocarbons (PAHs), for which maximum levels in food are set in the EU by the Commission Regulation (EU) 2023/915 and in Switzerland by the regulation SR 817.022.15. Therefore, a whey protein powder–based certified reference material (WP-CBR001) was developed according to the requirements of ISO 17034 and the recommendations of ISO Guide 35:2017 containing the four PAHs benz[a]anthracene (BaA), benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF), and chrysene (Chr). We show that the choice of solvent is of crucial importance to extract the PAHs completely from this matrix. Only polar and protic solvents such as methanol or water provided access for complete extraction of the PAHs. In contrast, nonpolar and polar aprotic solvents, such as n-hexane or ethyl acetate, exhibited only very low extraction efficiencies below 20%. The certified mass fractions and expanded uncertainties (k = 2) were (3.17 ± 0.32) µg/kg BaA, (4.18 ± 0.48) µg/kg BaP, (4.73 ± 0.49) µg/kg BbF, and (2.85 ± 0.33) µg/kg Chr. These values were verified by an interlaboratory comparison study and by the gravimetric mass fractions obtained from production data.Graphical abstract

Validation and interlaboratory comparison of anticoagulant rodenticide analysis in animal livers using ultra-performance liquid chromatography-mass spectrometry.

Anticoagulant rodenticides (ARs) are used to control rodent populations. Poisoning of non-target species can occur by accidental consumption of commercial formulations used for rodent control. A robust method for determining ARs in animal tissues is important for animal postmortem diagnostic and forensic purposes. We evaluated an ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) method to quantify 8 ARs (brodifacoum, bromadiolone, chlorophacinone, coumachlor, dicoumarol, difethialone, diphacinone, warfarin) in a wide range of animal (bovine, canine, chicken, equine, porcine) liver samples, including incurred samples. We further evaluated UPLC-MS in 2 interlaboratory comparison (ILC) studies; one an ILC exercise (ICE), the other a proficiency test (PT). The limits of detection of UPLC-MS were 0.3-3.1 ng/g, and the limits of quantification were 0.8-9.4 ng/g. The recoveries obtained using UPLC-MS were 90-115%, and relative SDs were 1.2-13% for each of the 8 ARs for the 50, 500, and 2,000 ng/g spiked liver samples. The overall accuracy from the laboratories participating in the 2 ILC studies (4 and 11 laboratories for ICE and PT studies, respectively) were 86-118%, with relative repeatability SDs of 3.7-11%, relative reproducibility SDs of 7.8-31.2%, and Horwitz ratio values of 0.5-1.5. Via the ILC studies, we verified the accuracy of UPLC-MS for AR analysis in liver matrices and demonstrated that ILC can be utilized to evaluate performance characteristics of analytical methods.

Transferability and reproducibility of exposed air-liquid interface co-culture lung models

BackgroundThe establishment of reliable and robust in vitro models for hazard assessment, a prerequisite for moving away from animal testing, requires the evaluation of model transferability and reproducibility. Lung models that can be exposed via the air, by means of an air-liquid interface (ALI) are promising in vitro models for evaluating the safety of nanomaterials (NMs) after inhalation exposure. We performed an inter-laboratory comparison study to evaluate the transferability and reproducibility of a lung model consisting of the human bronchial cell line Calu-3 as a monoculture and, to increase the physiologic relevance of the model, also as a co-culture with macrophages (either derived from the THP-1 monocyte cell line or from human blood monocytes). The lung model was exposed to NMs using the VITROCELL® Cloud12 system at physiologically relevant dose levels. ResultsOverall, the results of the 7 participating laboratories are quite similar. After exposing Calu-3 alone and Calu-3 co-cultures with macrophages, no effects of lipopolysaccharide (LPS), quartz (DQ12) or titanium dioxide (TiO2) NM-105 particles on the cell viability and barrier integrity were detected. LPS exposure induced moderate cytokine release in the Calu-3 monoculture, albeit not statistically significant in most labs. In the co-culture models, most laboratories showed that LPS can significantly induce cytokine release (IL-6, IL-8 and TNF-α). The exposure to quartz and TiO2 particles did not induce a statistically significant increase in cytokine release in both cell models probably due to our relatively low deposited doses, which were inspired by in vivo dose levels. The intra- and inter-laboratory comparison study indicated acceptable interlaboratory variation for cell viability/toxicity (WST-1, LDH) and transepithelial electrical resistance, and relatively high inter-laboratory variation for cytokine production. ConclusionThe transferability and reproducibility of a lung co-culture model and its exposure to aerosolized particles at the ALI were evaluated and recommendations were provided for performing inter-laboratory comparison studies. Although the results are promising, optimizations of the lung model (including more sensitive read-outs) and/or selection of higher deposited doses are needed to enhance its predictive value before it may be taken further towards a possible OECD guideline.

97 Transferability and Reproducibility of Exposed Air-Liquid Interface co-Culture Lung Models

Abstract The establishment of reliable and robust in vitro models for hazard assessment, moving away from animal testing, requires evaluation of model transferability and reproducibility. Lung models that can be exposed via the air, by means of an air-liquid interface are promising in vitro models for evaluating the safety of nanomaterials (NMs) after inhalation exposure. We performed an inter-laboratory comparison study to evaluate the transferability and reproducibility of a lung model consisting of the human bronchial cell line Calu-3 as monoculture and, to increase the relevance of the model, also as co-culture with macrophages (derived from the THP-1 monocyte cell line or from human blood monocytes). Exposure employed the VITROCELL® Cloud12 system at physiologically relevant doses. Overall, the results of the seven participating laboratories are quite similar. No effects of lipopolysaccharide (LPS), quartz (DQ12) or titanium dioxide (TiO2) NM-105 exposure on cell viability and barrier integrity were seen in the monoculture and co-culture models. Most laboratories showed that LPS induced cytokine release in the co-culture models, but not the monoculture model. Exposure to DQ12 or TiO2 did not increase cytokine release, probably due to too low deposited doses. Interlaboratory variability indicated acceptable variation for cell viability measurements and relatively high variation for cytokine measurements. Based on the transferability and reproducibility of the model and its air exposure we are confident that, based on detailed SOPs, performing a new round of interlaboratory comparison, and increasing the deposited doses of NMs, these models may be taken further towards a possible OECD guideline.

Interlaboratory comparison study of a chemical profiling method for methylphosphonic dichloride, a nerve agent precursor

An interlaboratory evaluation of a chemical profiling method for a nerve agent precursor was performed by eight chemical analytical laboratories with a wide global distribution. A set of chemical attribution signatures (i.e. impurities) present in methylphosphonic dichloride (DC) were analysed by gas chromatography-mass spectrometry (GC/MS). The GC/MS analyses were performed with the individual instrumentation and analytical methods used by the laboratories in proficiency tests organized by the Organisation for the Prohibition of Chemical Weapons (OPCW). Two batches of DC, produced by two different production routes, were dispatched together with one reference sample. By the use of a targeted MS-library, sixteen chemical attribution signatures were analysed in the DC samples. Retention indices, mass spectra and relative peak areas of the GC/MS data were evaluated. When the within batch-data of the eight laboratories were compared, similarity values of 0.720–0.995 were obtained. In addition, the two different batches had clearly unique chemical attribution profiles, as indicated by a large between batch-distance similarity values of 0.509–0.576. Retention indices showed less than ± 14 retention index unit variation for the 16 chemical attribution signatures. This study showed the potential of getting consistent chemical profiling data over multiple laboratories by the use of retention indices for alignment of GC/MS-data. The results indicate that the GC/MS instrumentation and methods used at most OPCW designated laboratories are valid tools for the acquisition of chemical profiling data of importance for the attribution analysis to study suspected production or use of chemical warfare agents.

Detection of adulterated meat products by a next-generation sequencing-based metabarcoding analysis within the framework of the operation OPSON X: a cooperative project of the German National Reference Centre for Authentic Food (NRZ-Authent) and the competent German food control authorities

The German National Reference Centre for Authentic Food (NRZ-Authent) and the competent German food control authorities of the federal states cooperated within the framework of the 10th joint Europol INTERPOL operation OPSON (OPSON X) in the detection of adulterated meat products. A total of 63 meat product samples were collected and analysed by the authorities using standard analytical procedures and subjected to a recently published 16S rDNA metabarcoding analysis. The sequence reads were analysed using 3 bioinformatics data processing strategies. The study aimed to gain additional data on the test samples regarding the authenticity of the declared species and to validate the 16S rDNA metabarcoding method with representative samples. The method was tested not only on 63 test samples, but also on 5 commercial samples from 2 interlaboratory comparison studies and 9 mock mixtures in parallel. The 16S rDNA metabarcoding method was able to detect species that were not target species of the used standard analytical methods, but failed, as shown previously, to detect fallow deer. Otherwise, the qualitative results of the 16S rDNA metabarcoding method were very similar to those of the methods currently in use by the German food control laboratories. Thus, the method has great potential to be used as a screening method for the authentication of mammal and poultry species in meat products.

Innovative reference materials for method validation in microplastic analysis including interlaboratory comparison exercises

Reference materials (RMs) are vital tools in the validation of methods used to detect environmental pollutants. Microplastics, a relatively new environmental pollutant, require a variety of complex approaches to address their presence in environmental samples. Both interlaboratory comparison (ILC) studies and RMs are essential to support the validation of methods used in microplastic analysis. Presented here are results of quality assurance and quality control (QA/QC) performed on two types of candidate microplastic RMs: dissolvable gelatin capsules and soda tablets. These RMs have been used to support numerous international ILC studies in recent years (2019–2022). Dissolvable capsules containing polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyethylene (PE), and polystyrene (PS), in different size fractions from 50 to 1000 µm, were produced for one ILC study, obtaining relative standard deviation (RSD) from 0 to 24%. The larger size fraction allowed for manual addition of particles to the capsules, yielding 0% error and 100% recovery during QA/QC. Dissolvable capsules were replaced by soda tablets in subsequent ILC studies and recovery test exercises because they were found to be a more reliable carrier for microplastic RMs. Batches of soda tablets were produced containing different single and multiple polymer mixtures, i.e., PE, PET, PS, PVC, polypropylene (PP), and polycarbonate (PC), with RSD ranging from 8 to 21%. Lastly, soda tablets consisting of a mixture of PE, PVC, and PS (125–355 µm) were produced and used for recovery testing during pretreatment of environmental samples. These had an RSD of 9%. Results showed that soda tablets and capsules containing microplastics >50 µm could be produced with sufficient precision for internal recovery tests and external ILC studies. Further work is required to optimize this method for smaller microplastics (< 50 µm) because variation was found to be too large during QA/QC. Nevertheless, this approach represents a valuable solution addressing many of the challenges associated with validating microplastic analytical methods.

Development of an equilibrium dialysis ID-UPLC-MS/MS candidate reference measurement procedure for free thyroxine in human serum

BackgroundAccurate and reliable measurement of human serum free thyroxine (FT4) is critical for the diagnosis and treatment of thyroid diseases. However, concerns have been raised regarding the performance of FT4 measurements in patient care. Centers for Disease Control and Prevention Clinical Standardization Programs (CDC-CSP) address these concerns by creating a FT4 standardization program to standardize FT4 measurements. The study aims to develop a highly accurate and precise candidate Reference Measurement Procedure (cRMP), as one key component of CDC-CSP, for standardization of FT4 measurements. MethodsSerum FT4 was separated from protein-bound thyroxine with equilibrium dialysis (ED) following the recommended conditions in the Clinical and Laboratory Standards Institute C45-A guideline and the published RMP [20,21,23]. FT4 in dialysate was directly quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. Gravimetric measurements of specimens and calibrator solutions, calibrator bracketing, isotope dilution, enhanced chromatographic resolution, and T4 specific mass transitions were used to ensure the accuracy, precision, and specificity of the cRMP. ResultsThe described cRMP agreed well with the established RMP and two other cRMPs in an interlaboratory comparison study. The mean biases of each method to the overall laboratory mean were within ±2.5%. The intra-day, inter-day, and total imprecision for the cRMP were within 4.4%. The limit of detection was 0.90 pmol/L, which was sufficiently sensitive to determine FT4 for patients with hypothyroidism. The structural analogs of T4 and endogenous components in dialysate did not interfere with the measurements. ConclusionOur ED-LC-MS/MS cRMP provides high accuracy, precision, specificity, and sensitivity for FT4 measurement. The cRMP can serve as a higher-order standard for establishing measurement traceability and provide an accuracy base for the standardization of FT4 assays.

Growth and characterization of gold nanoislands & quantum dots on marker-based SiO2/Si substrate for SEM resolution intercomparison studies

A scanning electron microscope (SEM) is the powerhouse tool of nanoscience & nanotechnology and the high resolution is one of the important parameters that decide the instrument’s quality. The reference materials (RM) like gold nanoislands or tin spheres on carbon substrate separated by a nanogap are often used to get the practically attainable resolution of the SEM instrument. Such RM contains millions of gold nanoislands and nanogaps but all of these RMs are non-traceable. Hence the practically obtained high resolution of an SEM remains doubtful unless and until intercompared with traceable RM. The development of a new SEM resolution test specimen sample with localized markers for the identification of a traceable critical dimension of a nanogap on a user-friendly SiO2/Si or Si3N4/Si chip is reported here. First, thin films of gold with thickness < 5 nm (ultra-thin film) and of about 10 nm (thin film) were deposited on a pre-patterned marker SiO2/Si chip to form the gold nanoparticles (Au NPs) by thermal annealing technique. The high-resolution SEM images show the formation of gold quantum dots (Au QDs) (average particle diameter <D> ∼ (18 ± 1) nm and average gap between two particles <S> ∼ (6 ± 1) nm) and gold nano-islands (Au NIs) (<D> ∼ (101 ± 3) nm and <S> ∼ (104 ± 2) nm). FESEM images show densely packed nano-islands exhibiting spherical geometry which is explained using dewetting mechanism followed by Ostwald ripening and coalescence resulting formation of Au QDs and Au NPs. Further, these marker-based resolution test chips can be used in inter or intra-comparison studies due to pre-patterned markers. First-time development of QDs on marker chip gives access to a particular nanogap among the millions of nanogaps present on the substrate, this type of RM will be used for inter-laboratory comparison studies in the future.

Determination of inorganic As, DMA and MMA in marine and terrestrial tissue samples: a consensus extraction approach

Environmental context Arsenic can be found in all environmental compartments in a large number of chemical forms of varying toxicity. We performed an inter-laboratory comparison study focusing on quantitation of some of the most toxic arsenic forms in seven different biological materials and found very good agreement among the submitted results. Certification of the studied materials will provide suitable quality control samples for environmentally relevant concentrations of arsenic in food products and biota.Rationale Arsenic (As) speciation analysis in biological matrices has been performed for several decades; however, there are very few matrix certified reference materials available for the validation of analytical methods. The literature data on the mass fractions of As species in the existing certified reference materials are inconsistent and suggest method extraction dependency.Methodology In the present study, an international round-robin study was organised to identify the possible sources of discrepancies in quantitation of several As species in different matrices of biological reference materials: one plant tissue, three marine and three terrestrial biological tissues. Each participating laboratory was provided with a set of identical calibration standard solutions, and analysed the samples by following a common group extraction method as well as using an in-house protocol.Results The results showed that significant biases can be introduced by insufficient verification of the analyte’s mass fractions in the calibration standard solutions. The choice of extraction method seems to have very little impact on the quantitation of As species in the studied plant and terrestrial biological tissues. However, following a prescribed extraction method led to significant reduction of uncertainties in more complex samples such as marine animal tissues.Discussion Some differences in the mass fractions of As species extracted from marine animal tissues in water (with and without H2O2) were observed and should be further investigated. Despite the variety of extraction methods used, very good agreement between reported mass fractions was achieved and the combined consensus values will be published as certified reference values of As species in the studied materials.

Photocatalytic activity of nanoparticles: the development of the standardized measurement for physiological conditions

Recently a new International Standard for testing nanomaterial photocatalytic activity under physiological conditions was issued by Technical Committee 229 (Nanotechnologies) of the International Organization for Standardization (ISO 20814:2019 Nanotechnologies-Testing the photocatalytic activity of nanoparticles for NADH oxidation). The document offers a robust, high throughput photocatalytic assay using a bio-compatible indicator nicotinamide amide dinucleotide (NAD) and provides a screening tool to gauge nanomaterial potency for phototoxicity. This paper describes the measurement principles behind this assay, the scope of the standard and its validation through an interlaboratory comparison study using a traceable standard reference material (SRM 1898).

Pilot Inter-Laboratory Comparison Study of Optical Genome Mapping Analysis for Cytogenomic Characterization of Hematological Malignancies: A Spanish Multicentric Study

Introduction. Optical genome mapping (OGM, Bionano Genomics) is a novel whole genome approach based on the imaging of long DNA molecules that allows detecting structural and copy number variants (CNV) at a high resolution. Recent publications in several hematologic malignancies have demonstrated the good performance of OGM to identify clinically relevant genetic aberrations, otherwise found by karyotyping, FISH and/or genomic microarrays. Despite its high potential as a diagnostic tool, consensus guidelines for OGM implementation in the clinical setting are still lacking. The aims of the study were: (1) To describe the inter-laboratory reproducibility in the identification and reporting of clinically relevant cytogenomic variants by OGM; (2) To outline the main causes of discrepancies among centers, to be addressed in future OGM recommendations. Patients and Methods. Six Spanish centers participated in this pilot inter-laboratory comparison. A total of 12 patients were included, analyzing three centers each. The analyzed samples comprised tumoral specimens from two chronic lymphocytic leukemia (CLL), one follicular lymphoma (FL), two multiple myeloma (MM), two primary myelofibrosis (PMF), one acute myeloid leukemia (AML), two adult T-cell acute lymphoblastic leukemia (T-ALL) and two pediatric B-cell acute lymphoblastic leukemia (B-ALL). High molecular weight DNA from peripheral blood (n=6) or bone marrow (n=6) samples was labelled, processed and imaged in a Saphyr System (Bionano Genomics). Molecule files from the rare variant analysis (obtained from the Bionano Access software) were shared among hospitals and each center analyzed the samples following the manufacturer's filtering/analysis recommendations. The concordance rate was calculated taking into account those variants included in the final curated variant list and those that would be included in the clinical report. Results. Globally, 152 variants were recorded by at least one center (median: 11/sample, range: 6-22), being 100 CNV, 44 translocations and 8 small insertions or inversions. Among them, 98 (68%) were considered clinically relevant as they were classified as pathogenic (disease-related or CNV >5Mb, n=62) or likely pathogenic (CNV<5Mb associated with translocations or translocations associated with pathogenic CNV, n=36). The remaining 54 were classified as variants of unknown significance (VUS). Regarding the detection of pathogenic and likely-pathogenic variants, the global concordance among centers was 83%, being 100% in 8/12 cases, around 90% in 3/12 cases and only 33% in one case. The latter had a low tumor burden and one laboratory did not perform a visual revision of the whole genome data (Figure). Remarkably, except for this case, 100% of concordance was obtained in the detection of the diagnostic and prognostic abnormalities described in guidelines (Table). Similar concordance rates were obtained for rearrangements (79%) and CNV gains and losses (86% and 85%, respectively). Concerning the inclusion in the clinical reports of those variants detected by all the centers, 94% (75/80) were concordantly reported. Discordances in the reporting included 3 pathogenic variants (one complex rearrangement and 2 losses with a low infiltration) and 2 likely pathogenic variants (complex rearrangements associated with CNV). In contrast, only 6/54 VUS (11%) were concordantly identified (3 CNV <500Kb and 3 translocations) and none of them was reported by all the centers. Conclusions. 1. The inter-laboratory concordance for the identification of the clinically relevant (diagnostic and/or prognostic) abnormalities was optimal. 2. Nevertheless, certain potential pathogenic aberrations were discordantly classified and/or reported; discrepancies were explained by a variable previous knowledge on the Bionano Access software, different filtering/analysis criteria and diverse professional expertise; 3. Future OGM international guidelines are needed to harmonize the introduction of such cytogenomic results in the clinical reports for hematological malignancies. Acknowledgements: 2017SGR437 and 2017SGR288 and CERCA Programme, Generalitat de Catalunya; Fundació Internacional Josep Carreras; PI19/01828 and PI21/00022 from ISCIII; Premios FIHGUV 20/425; SEHH (C. G-G personal grant) and ACIF/2021/169, from Generalitat Valenciana (R.G-S predoctoral contract). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal