In-house Reference Research Articles

A novel method for high-precision triple oxygen isotope analysis of diverse Earth materials using high temperature conversion–methanation–fluorination and isotope ratio mass spectrometry

Triple oxygen isotope geochemistry is a growing field that investigates mass dependent and mass independent fractionation in 17O/16O relative to 18O/16O imparted by geochemical, physical, and biological processes. Many applications require 10 ppm-level precision in measurement of the Δ′17O parameter. Such analytical precisions are rarely realized for some materials (e.g. sulfates), and no method presently exists for many organic compounds. We describe a new analytical method that combines the versatility of high-temperature conversion (HTC, also known as Temperature Conversion Elemental Analysis, TC/EA) with the high-precision of the methanation-fluorination method described by Passey et al. (2014). In this method, materials including organics, phosphates, sulfates, nitrates, carbonates, and silicates undergo high-temperature conversion of their oxygen to CO in a glassy carbon reactor at temperatures >1400 °C. This CO is then reacted with hydrogen at 560 °C over an iron catalyst to transfer CO oxygen to oxygen in H2O (a methanation reaction). This H2O is subsequently fluorinated by CoF3 to yield O2, which is then analyzed by mass spectrometry to determine δ17O and δ18O. Sample oxygen conversion is quantitative (or nearly so) for most compatible materials, resulting in excellent reproducibility in Δ′17O (<10 ppm 1σ) and no clear evidence of fractionation affecting Δ′17O. We report the first (to our knowledge) high-precision Δ′17O measurements for organics, including benzoic acid (IAEA-601, IAEA-602), cellulose (IAEA-CH-3), keratin (USGS KHS), and honey (USGS 83). We present results for silver phosphates (NBS 120c, USGS 80, USGS 81, B2207), carbonates (IAEA-603, IAEA-C1, NBS 18), sulfates (IAEA-SO-5, IAEA-SO-6), a silicate (NBS 28), and waters (VSMOW2, SLAP2). We also present results from several in-house reference materials, including a silver nitrate and a phosphate that was equilibrated with isotopically exotic waters. Analysis of carbonates, sulfates, and silicates requires accelerants (e.g., graphite and AgCl or KF) to achieve high yields and ppm-level precision in Δ′17O. Waters can be analyzed using the same method during the same analytical sessions as solid materials, making determination of water-mineral fractionation factors straightforward and enabling values to be placed directly on the VSMOW-SLAP scale by concurrent analyses with those reference materials. We find that the method is especially well-suited for organics, sulfates, phosphates, and nitrates, however traditional fluorination methods may be better suited for many silicate minerals.

The development and establishment of a heat inactivated preparation of Mycobacterium tuberculosis (H37Rv) as the first international standard for nucleic acid amplification techniques

A need to develop an inactivated Mycobacterium tuberculosis (Mtb) preparation, to be used as a DNA standard, as an important and urgent requirement for Tuberculosis (TB) diagnostics standardization was identified in 2018. A candidate material was generated using a heat inactivated culture of Mtb strain H37Rv. A lyophilised preparation was evaluated for its suitability as an International Standard for molecular detection of Mtb DNA in an international collaborative study. Together with the use of quantitative PCR assays and rapid diagnostic tests, this candidate standard was demonstrated to be fit-for-purpose. Based on the results from this collaborative study, it is proposed this lyophilised heat inactivated Mtb preparation (NIBSC code: 20/152) to be established by the World Health Organization Expert Committee on Biological Standardization, as the First WHO International Standard for Mycobacterium tuberculosis (H37Rv) for nucleic acid amplification techniques with an assigned unitage of 6.3 log10 or 2 × 106 International Units per vial.The intended uses of this IS are for calibration of secondary or in-house reference preparations used in the assays for the molecular detection of Mtb DNA. It may also be used for assay validation and monitoring the performance in terms of limit of detection of rapid diagnostic tests.

Precision geochemical (ICP-MS) analysis of Precambrian rocks: the method and accuracy estimation

The article discusses the results of measuring the composition of Early Precambrian mafic rocks and granitoids of the eastern part of the Fennoscandian Shield by inductively coupled plasma mass spectrometric analysis (ICP-MS). It is demonstrated that the bul kprobe sample composition can be determined both by acid digestion of samples in an open system and by autoclave digestion. The accuracy and reproducibility of the analysis were monitored by analyzing the international and Russian standards and the in-house reference sample: BHVO-2, SHD-2A, GSP-2, C-1412

Evaluation of alternative gas chromatographic and mass spectrometric behaviour of trimethylsilyl-derivatives of non-hydrolysed sulfated anabolic steroids.

Sulfated metabolites have shown to have potential as long-term markers (LTMs) of anabolic-androgenic steroid (AAS) abuse. The compatibility of gas chromatography-mass spectrometry (GC-MS) with trimethylsilyl (TMS)-derivatives of non-hydrolysed sulfated steroids has been demonstrated, where, after derivatisation, generally, two closely eluting isomers are formed that both have the same molecular ion [M-H2 SO4 ]•+ . Sulfated reference standards are in limited commercial availability, and therefore, the current knowledge of the GC-MS behaviour of these compounds is mainly based on sulfating and analysing the available standard reference material. This procedure can unfortunately not cover all of the current known LTMs as these are often not available as pure substance. Therefore, in theory, some metabolites could be missed as they exhibit alternative behaviour. To investigate the matter, in-house sulfated reference materials that bear resemblance to known sulfated LTMs were analysed on GC-MS in their TMS-derivatised non-hydrolysed state. The (alternative) gas chromatographic and mass spectrometric behaviour was mapped, evaluated and linked to the corresponding steroid structures. Afterwards, using fraction collection, known sulfated LTMs were isolated from excretion urine to confirm the observed findings. The categories that were selected were mono-hydroxy-diones, 17-methyl-3,17-diols and 17-keto-3,16-diols as these are commonly encountered AAS conformations. The ability to predict the GC-MS behaviour of non-hydrolysed sulfated AAS metabolites is the corner stone of finding new metabolites. This knowledge is also essential, for example, for understanding AAS detection analyses, for the mass spectrometric characterization of metabolites of new designer steroids or when one needs to characterize an unknown steroid structure.

Developing Methods for Maintaining Genetic Diversity in Novel Aquaculture Species: The Case of Seriola lalandi.

Developing sound breeding programs for aquaculture species may be challenging when matings cannot be controlled due to communal spawning. We developed a genotyping-by-sequencing marker panel of 300 SNPs for parentage testing and sex determination by using data from an in-house reference genome as well as a 90 K SNP genotyping array based on different populations of yellowtail kingfish (Seriola lalandi). The minimum and maximum distance between adjacent marker pairs were 0.7 Mb and 13 Mb, respectively, with an average marker spacing of 2 Mb. Weak evidence of the linkage disequilibrium between adjacent marker pairs was found. The results showed high panel performance for parental assignment, with probability exclusion values equaling 1. The rate of false positives when using cross-population data was null. A skewed distribution of genetic contributions by dominant females was observed, thus increasing the risk of higher rates of inbreeding in subsequent captive generations when no parentage data are used. All these results are discussed in the context of breeding program design, using this marker panel to increase the sustainability of this aquaculture resource.

Examination of amino acid hydrogen isotope measurements of scalp hair for region-of-origin studies.

Hydrogen isotope (δ2 H) analysis of keratinaceous bulk tissues has been used in forensic science to reconstruct an individual's travel history or determine their region-of-origin. Here, we use a compound-specific approach to examine patterns of individual amino acid δ2 H values in relation to those of local tap water, bulk scalp hair tissues, and region-of-origin. We measured δ2 H values of amino acids in anonymously collected scalp hair (n = 67) and tap water from 28 locations in the United States. Samples were hydrolyzed into their constituent amino acids, derivatized alongside in-house reference materials, and analyzed in triplicate using a GC-C-IRMS system. Non-essential amino acid (AANESS ) δ2 H values and their corresponding tap water samples varied systematically across continental regions. Hydrogen isotope values of alanine, glutamic acid, and glycine were significantly correlated with tap water and an estimated 42%-51% of the hydrogen atoms in these AANESS were derived from tap water. We used linear discriminate analysis (LDA) to explore regional patterns in scalp hair bulk tissue and amino acid δ2 H values. For the model that included AANESS data, 87% of the variance was explained by the first linear discriminant axis (LD1), and was driven by bulk hair tissue, alanine, and proline. This model had an overall 72% successful reclassification with samples from the south and northwest regions reclassifying correctly 92% and 78% of the time, respectively. For the model that included AAESS data, LD1 explained 81% of the variation and was driven bulk hair, threonine, valine, phenylalanine, and isoleucine. The overall reclassification rate for the model that included AAESS was 70%. Our findings suggest that δ2 H analyses of AANESS and AAESS could help improve geolocation models for human and wildlife forensics by simultaneously providing information about both dietary and tap water inputs of hydrogen to tissue synthesis.

Development of In-House Reference Materials for the Composition of Soils Polluted With Heavy Metals

Pollution of agricultural soils with heavy metals is an acute problem both in Russia and globally. The use of standard methods for soil monitoring is hampered by the need to regularly modernize monitoring equipment. Therefore, the development of matrix multicomponent reference materials (RM) for the composition of soils contaminated with heavy metals in concentrations exceeding their limiting values in natural soils is a highly relevant research task.To develop in-house multicomponent RMs of soil composition with the certified value of mobile forms of copper, zinc, lead, cadmium, nickel, cobalt and manganese in concentrations exceeding their trace levels.Soils for the development of RMs were collected in residential areas of the Vladimir Oblast and Chelyabinsk Oblast subjected to technogenic pollution. Samples were prepared according to a conventional methodology. The metrological characteristics of the developed RMs were determined by the method of interlaboratory comparison with the participation of 59 accredited testing laboratories. The mobile forms of heavy metals were determined by the atomic absorption method. An assessment of the error and uncertainty of the certified RM value was performed.Two in-house RMs of the composition of soils containing mobile forms of copper, zinc, lead, cadmium, nickel, cobalt and manganese at the level of tens of maximum allowable concentrations were developed. The developed RMs were found to be homogeneous in terms of all certified indicators. The calculated error did not exceed admissible values. The shelf life of RM samples was determined to be two years.The developed in-house RMs can be used for supporting agroecological monitoring and research programmes.

Combined Inhibition of CDK2 and BCL2 Overrides Resistance to Targeting BTK and CDK4/6 in Mantle Cell Lymphoma Therapy

Mantle cell lymphoma (MCL) remains incurable due to the development of drug resistance, despite the plethora of therapies available. Each successive treatment failure is associated with a more rapidly proliferating disease and fewer treatment options. Understanding the drug-resistance mechanism and developing effective, well-tolerated, new therapies for MCL are urgently needed. Unrestrained proliferation of MCL cells is mainly driven by aberrant cyclin D1 expression and dysregulated CDK4 activity that promotes cell cycle progression from G1 to S. In our phase 1 clinical trial inhibiting CDK4/6 with palbociclib and BTK with ibrutinib (PALIBR) in recurrent MCL, the complete response rate (CR) was 42% compared to 21% in response to ibrutinib alone, despite a comparable 67% overall response rate. Moreover, 5 patients remain on therapy (2 CR and 3 PR) for ~ 8 years, suggesting that CDK4/6 inhibition deepens and prolongs the clinical response to BTK inhibition in MCL. However, resistance emerges in some patients. To elucidate the underpinnings, we undertook integrative longitudinal genomic analysis of sequential tissue and blood specimens (n=53) of 27 MCL patients before, during therapy and on progression, and 4 treatment-naïve MCL patients and 4 normal subjects as controls. Whole exome and whole transcriptome sequencing of freshly isolated MCL cells and B cells revealed that resistance to PALIBR is rooted in homozygous deletion of RB1 (direct target of CDK4/6) or compound copy number variation (cCNV) - concurrent hemizygous loss of RB1 and CDKN2A (p16, CDK4/6 inhibitor) and gain of CDK4. In other resistant patients, the RB protein is silenced by translational termination mutation. Single-cell RNA-sequencing (scRNA-seq, 210,000 cells) of corresponding PBMCs using a unique in-house MCL RNA reference library reveals that MCL cells comprise 4 transcriptomically distinct clusters. Cluster 1 (C1) is similar to quiescent normal B cells; C2 resembles hyper-activated B cells enriched for signatures of BCR and cytokine signaling; C3 represents non-proliferating, long-lived MCL cells; and C4 is highly proliferative, expanding with disease progression. RNA velocity analysis further uncovered that C2 fuels the transition to C4 with disease progression, whereas C3 is a terminal node (Fig 1). In C2 and C3 MCL cells, CDK2 is highly elevated, indicating evasion of CDK4/6 inhibition, and PIK3IP1 (negative PI3K regulator) is silenced, suggesting that compensatory PI3K activation in BTKi resistance (Chiron et al, Cancer Discovery 2014) is secondary to loss of cell cycle control. BCL2 is markedly elevated in C2, greater in C3 MCL cells along with increases in anti-apoptotic BCL2L1(BCL-XL) and BCL-2A1 and decreases in pro-apoptotic PMAIP1 (NOXA) expression. This suggests combined CDK2 and BCL2 inhibition as a rational approach to override CDK4/6i and BTKi resistance. Indeed, combined inhibition of CDK2 with PF-07104091 and BCL2 with venetoclax (PF-VEN) cooperatively impaired growth and killed ibrutinib-resistant MAVER-1 cells, more effectively in the isogenic derivative MAVER-1R cells which we generated to recapitulate cCNV in PALIBR resistance - depletion of RB protein through translational termination mutation (W99, AAF 75%) in RB1, loss of CDKN2A and gain of CDK4. Mechanistically, MAVER-1R cells are accelerated in G1 to S progression, based on elevation of CDK2 and TK1 expression and cell cycle profiling. They are intrinsically apoptotic and vulnerable to BCL2 inhibition due to a marked reduction in cells expressing anti-apoptotic BCL2L1 and IRF4, and universal expression of pro-apoptotic PMAIP1 (NOXA). Importantly, combined CDK2 and BCL2 inhibition synergistically kills MCL cells of a patient resistant to PALIBR ex vivo. In summary, by longitudinal genomic analysis, we have provided the first evidence that 1) resistance to CDK4/6i and BTKi stems from MCL intrinsic cCNV; 2) MCL cells comprise 4 major transcriptomically distinct clusters; cluster 2 expressing copious CDK2 and BCL2 is pivotal in fueling proliferation of MCL cells in resistance; 3) cluster 3 cells also accumulate with resistance due to longevity; 4) combined inhibition of CDK2 and BCL2 overrides resistance to CDK4i and BTKi, in MCL cell lines and primary MCL cells from PALIBR resistant patient. Combined inhibition of CDK2 and BCL2, therefore, represents a mechanism-based strategy to overcome CDK4/6i and BTKi resistance in MCL therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Are the resources provided by Business Incubators suitable to the needs of their customers?

Highlights: Studies on business incubation reveal that little is known about what incubatees really want and need. What are their challenges? Are the available resources adequate to meet them? This work attempts to find methods to assist in answering these questions. Goal: The present study investigates the suitability of resources supplied by incubators to the needs of incubated firms with the goal of proposing a methodology to assess resources’ customer satisfaction and identify opportunities for satisfaction enhancement. Design/Methodology/Approach: The methodology integrates the research on Models and Typologies of incubators with the Resource-Based View and adopts a comparative customer-supplier framework aiming to enable a systemic approach to customer satisfaction. For validation purposes, empirical investigation was conducted in an incubator located in the metropolitan region of Rio de Janeiro, Brazil. Field investigation adopted mixed methods through semi-structured interviews and convenience sample selection. Results: The empirical testing shows the methodology meets its purposes. Findings on customer satisfaction indicate three clear opportunities towards its enhancement. Limitations of the investigation: The methodology was validated in one incubator with small population and sample. Practical Implications: The methodology applied to a single incubator provides in-house reference to satisfaction enhancement. Aiming further-reaching initiatives, the methodology should be evaluated on larger samples and tailored to address cross-incubator comparison. Originality/ Value: The method combines two stablished theoretical strands on incubation with a comparative customer-supplier approach. The resultant framework enables a systemic approach to customer satisfaction grounded in a set of field survey-collected information.

Preparation of in-house reference material of Fe, Ca, Ni and Si in carbon sample from an aluminum industry and results of a proficiency test

This work presents the preparation of a reference material for Fe, Ca, Ni and Si in order to ensure the quality of the analysis of these elements in routine samples of carbonaceous materials in a aluminum industry. The sample was fractioned and bottled for homogeneity and stability studies before being shipped to interlaboratory certification. Then, we sent a bottle for each one of seven participating laboratories to define the reference values. The results of concentration for Fe, Ca, Ni and Si, after outlier exclusion were 589.1 ± 25.3, 142.9 ± 12.0, 178.1 ± 9.3 and 134.2 ± 6.6, respectively. Finally, we applied the technique of z-score, to evaluate the performance of the participating laboratories. After the results of this study we can conclude that the carbon material can be used like reference material for in-house control of Fe, Ca, Ni and Si analyses.

Hyperspectral imaging solutions for the non-invasive detection and automated mapping of copper trihydroxychlorides in ancient bronze

Ancient bronze is subject to complex degradation which can lead, in cases where copper chlorides are present, to a cyclic and self-sustaining degradation process commonly referred to as “bronze disease”. If left untreated, bronze disease can eat away at a bronze object until it is entirely deteriorated. The presence of copper trihydroxychlorides is indicative that this process is underway and therefore the detection of these corrosion products is necessary in guiding conservation of ancient bronze artefacts. In this paper we present a high spatial/spectral resolution short wave infrared (SWIR) imaging solution for mapping copper trihydroxychlorides in ancient bronze, combining hyperspectral imaging with an in-house developed unsupervised machine learning algorithm for automated spectral clustering. For this work, verification was obtained through use of an in-house developed reference database of typical ancient bronze corrosion products from several archaeological sites, and from collections of the National Museum of China. This paper also explores the suitability, and limitations, of a visible to near-infrared (VNIR) hyperspectral imaging system as a more accessible solution for mapping copper trihydroxychlorides associated with bronze disease. We suggest that our hyperspectral imaging solution can provide a non-invasive, rapid, and high resolution material mapping within and across bronze objects, particularly beneficial for analysing large collections in a museum setting.

Quantitative mapping of mercury and selenium in mushroom fruit bodies with laser ablation–inductively coupled plasma-mass spectrometry

This work describes the development of a novel method for quantitative mapping of Hg and Se in mushroom fruit body tissues with laser ablation coupled to inductively coupled plasma-mass spectrometry (LA-ICP-MS). Different parameters of the protocol for preparation of the standards used for quantification via external calibration were assessed, e.g., the dissolution temperature of gelatin standards and the addition of chitosan and l-cysteine as additives to the gelatin-based calibration droplets to better match the sample matrix. While chitosan was not suited for this purpose, the presence of l-cysteine considerably improved the figures of merit of the calibration, leading to limits of detection of 0.006 and 0.3 µg g−1 for Hg and Se, respectively, at a pixel size of 20 × 20 µm. Further, an in-house reference material, ideally suited for the validation of the method for application to mushroom samples, was successfully prepared from a paste of Boletus edulis. The newly developed method was used to investigate the distribution of Hg and Se in tissue sections of five porcini mushroom individuals of three different species (Boletus edulis, Boletus aereus, and Boletus pinophilus) and one sample of a parasol mushroom (Macrolepiota procera). For one sample, additional areas were ablated at higher spatial resolution, with a laser spot size down to 5 µm, which allows a detailed investigation of the spatial distribution of Hg and Se in mushrooms.Graphical abstract

Analysis and Risk Assessment of Insulin-Potentiating Elements in Addua (Balanites Aegyptiaca) Leaf, Seed and Bark.

Purpose: This study investigated certain trace elements having therapeutic properties related to diabetes mellitus and to look into the health risk assessment of the elements. The investigated samples were the bark, leaf and seed of Balanites aegyptiaca, desert date (Addua). These plants are traditionally used in the management of diabetes in Eritrea, Egypt, and Ethiopia.&#x0D; Methodology: The elemental analysis was conducted using Flame Emission Photometer and Atomic Absorption Spectroscopy (AAS) techniques. The accuracy of the methods was verified using in-house reference materials (CRMs) and no significant differences were observed between the measured and certified values.&#x0D; Findings: The analysis displayed variable concentrations of the different trace elements found to be in the permissible limit defined by WHO. The metals are in the order of decreasing concentrations in the bark sample K &gt; Ca &gt; Mg &gt; Co &gt; Fe &gt; Cu &gt; Zn &gt; Mn. In the leaf, the ranking order is K &gt; Ca &gt; Mg &gt; Fe &gt; Zn &gt; Mn. The seed sample appeared in the order K &gt; Ca &gt; Mg &gt; Fe &gt; Zn &gt; Mn. The THQ for the non-carcinogenic risk were all &lt; 1 for all metals and the various exposure pathways. The trend is same for HI for both children and adults. This is a pointer to the fact that no immediate danger due to presence of potential carcinogens. Furthermore, the study revealed the order of pathway in the following decreasing order HIingestion &gt; HIdermal &gt; HIinhalation and most of all, the HI values for adults generally higher than that of the children. However, this suggests that adults are at greater risks of and could be more susceptible to potential carcinogenic risk from continual exposure.&#x0D; Recommendation: The findings calls for concern on the regular use of some of the infusion from this plant.

Mass Difference Matching Unfolds Hidden Molecular Structures of Dissolved Organic Matter.

Ultrahigh-resolution Fourier transform mass spectrometry (FTMS) has revealed unprecedented details of natural complex mixtures such as dissolved organic matter (DOM) on a molecular formula level, but we lack approaches to access the underlying structural complexity. We here explore the hypothesis that every DOM precursor ion is potentially linked with all emerging product ions in FTMS2 experiments. The resulting mass difference (Δm) matrix is deconvoluted to isolate individual precursor ion Δm profiles and matched with structural information, which was derived from 42 Δm features from 14 in-house reference compounds and a global set of 11 477 Δm features with assigned structure specificities, using a dataset of ∼18 000 unique structures. We show that Δm matching is highly sensitive in predicting potential precursor ion identities in terms of molecular and structural composition. Additionally, the approach identified unresolved precursor ions and missing elements in molecular formula annotation (P, Cl, F). Our study provides first results on how Δm matching refines structural annotations in van Krevelen space but simultaneously demonstrates the wide overlap between potential structural classes. We show that this effect is likely driven by chemodiversity and offers an explanation for the observed ubiquitous presence of molecules in the center of the van Krevelen space. Our promising first results suggest that Δm matching can both unfold the structural information encrypted in DOM and assess the quality of FTMS-derived molecular formulas of complex mixtures in general.

Abstract 5116: Analytical performance of ELSA-seq, a blood-based test for early detection of multiple cancers

Abstract Introduction: Detection of cancer is its early stages can potentially improve therapeutic effectiveness. Previously, we demonstrated that ELSA-seq, a machine-learning-aided methylation profiling test, can detect and locate multiple cancers with high accuracy in plasma samples. Here, we evaluated the analytical performance of a refined test version of ELSA-seq, including analytical sensitivity, specificity, repeatability/reproducibility, and robustness. Methods: The classification algorithm and cut-offs for the ELSA-seq test were established in the THUNDER study (NCT04820868). Here, we describe the analytical performance using both plasma samples from the THUNDER study and DNA blends from cell lines. Analytical sensitivity was established by defining the limit of detection (LoD) using in-house cfDNA Methylation Reference Standards (MRS) with lowest DNA input for the assay. In brief, fragmented genomic control DNA (NA24385, Coriell Institute) was used as a diluent to contrive human cancer cell lines with defined mixing ratios (tumor fractions), which was further verified by digital droplet PCR (ddPCR). The LoD was determined by the lowest tumor fractions at which accurate DOC and TOO was reported in at least 95% of replicates. As variant allele frequency (VAF) is widely used as a surrogate measurement for tumour fractions, we also used ultra-deep mutation sequencing to attain VAF as an independent piece of evidence. Analytical specificity was assessed by the true negative rate in 120 plasma samples from age-matched healthy donors. A batch-to-batch repeatability/reproducibility study was carried out using 168 clinical samples processed across multiple reagent lots, instruments, and operators. Robustness was evaluated using common interfering substances that potentially could be present in plasma samples such as hemoglobin, bilirubin, triglycerides, and genomic DNA. Testing was performed using 24 cancer and 24 non-cancer samples, with or without interfering substances. Results: At 5ng input mass (approximating from 1ml plasma), the LOD95 was estimated down to 0.05% (tumor fractions) or 0.02% (VAF) among 6 cancer cell lines. 2 false positives were detected in 120 age-matched healthy donor samples, yielding a specificity of 98.3% (95%CI: 93.5-99.7%). All test results were concordant across multiple reagent lots, instruments, and operators (100% repeatability and reproducibility), and high Pearson correlation coefficients (&amp;gt;99%) were observed in the pair-wise comparison. In addition, none of the substances tested interfered with the assay. Conclusions: These results suggest that ELSA-seq is a highly sensitive test for detection of the trace amount of tumor-derived methylation signals in plasma samples. The performance is highly reproducible and robust, which is critical for clinical implementations. Citation Format: Bingsi Li, Jing Su, Guangliang Zhang, Jiayue Xu, Jianlong Peng, Ya Zhou, Fujun Qiu, Shuai Fang, Xiaofang Wen, Guoqiang Wang, Jing Zhao, Hao Wang, Shangli Cai, Zhihong Zhang. Analytical performance of ELSA-seq, a blood-based test for early detection of multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5116.

Abstract 4104: Low-cost, simple and rapid assay for single-molecule detection of gene fusions from RNA with ASPYRE technology

Abstract Introduction: The FDA has approved 11 therapies for non-small cell lung cancer (NSCLC) patients diagnosed with gene fusions. Importantly, these life-saving targeted therapies remain underutilized due to a lack of cost-effective, comprehensive, and simple-to-use technologies for gene fusion detection. Our previous work using ASPYRE technology (allele-specific pyrophosphorolysis reaction; Silva et al, 2020) demonstrated detection of somatic variants at single-molecule level from DNA extracted from tumor tissue. However, DNA-based fusion detection requires tiling across large introns, which limits sensitivity particularly if breakpoints are located in repetitive regions of the genome. Here, we show that ASPYRE technology can also be utilized for highly sensitive detection of RNA fusions, scaling to over 30 fusion targets covering the main 3’ fusion partners (ALK, ROS1, RET, NTRK1, NTRK3) and from a single reverse transcription PCR (RT-PCR) amplification reaction. Methods: The ASPYRE assay consists of four sequential enzymatic stages: RT-PCR; enzymatic clean-up; combined exonuclease digestion, hybridization, pyrophosphorolysis and ligation; rolling circle isothermal amplification and detection. All targets are detected across two reaction wells using four channels on a standard real-time PCR instrument. A panel of synthetic RNA oligonucleotides comprising gene fusion boundaries was spiked into a background pool of lung-RNA, quantified using digital PCR (dPCR) and compared against a commercial reference standard to validate its suitability for assessing assay performance. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) lung tissue from healthy donors to determine assay specificity, and from FFPE lung tissue from NSCLC patients with known fusions to exemplify end-to-end sample analysis. Results: We demonstrate equivalence of our in-house reference samples to commercial reference standards. Using serial dilutions of input RNA standards, we show that detection by ASPYRE is consistent with detection of single molecules as input for common ROS1, ALK, RET and NTRK3 fusions. In addition, ASPYRE was able to detect previously confirmed ROS1 and ALK fusions and MET exon skipping variants in FFPE samples. ASPYRE had high specificity, correctly confirming the absence of RNA fusions and exon skipping variants in healthy control FFPE samples. Conclusions: ASPYRE approached single-molecule sensitivity for over 30 RNA fusion targets in a single reaction. The assay required only 1 ng RNA input, with a turn-around time of &amp;lt;4 hours from extracted RNA to result, opening the door to routine detection of RNA fusions from limited specimens. The simplicity and ease of use of ASPYRE, combined with its rapid turnaround time and analytical performance characteristics, provides a new tool for rapid and comprehensive treatment selection in NSCLC. Citation Format: Eleanor Gray, Justyna Mordaka, Efthimia Christoforou, Kristine von Bargen, Nicola Potts, Rebecca Palmer, Barnaby Balmforth. Low-cost, simple and rapid assay for single-molecule detection of gene fusions from RNA with ASPYRE technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4104.

High-throughput UPLC-Q-TOF-MS/MS coupled with multivariable data processing approach for the rapid screening and characterization of chemical constituents and potential bioactive compounds from DangguiShaoyao San.

Danggui Shaoyao San (DSS), a herbal formula, has been widely used for decades in China to treat senile dementia and dysmenorrhea. Here, an integrative high-throughput UPLC-Q-TOF-MS/MS method coupled with a multivariable data processing approach was established for rapidly screening and identifying chemical constituents and potential bioactive compounds from DSS. Through the comparison with mass fragment ions, relevant literature, and in-house reference material database coupled with MS cleavage mechanism, 150 chemical constituents, mainly including triterpenoids, flavonoids, phathalides, and organic acids, were tentatively characterized. Most of them were identified for the first time. Then, principal component analysis was used to evaluate the differences in chemical profiles between groups, whereas the variable importance of the projection (VIP) spectrum (VIP > 1) and the trend plot of orthogonal partial least squares discriminant analysis were applied to intuitively screen the candidate variables present only in the dosed group. Consequently, by comparison with all the characterized components in vitro, 23 potential bioactive compounds were successfully identified, comprising 5 triterpenoids, 4 phathalides, 4 flavonoids, 4 organic acids, 3 lactones, and 3 other compounds, which were present in various medicinal materials, reflecting a synergistic mechanism. This work developed a rapid, reliable, and robust approach for comprehensive characterization of the chemical components and potential bioactive compounds of DSS, providing solid data for further research on pharmacodynamic substances and pharmacological mechanisms of DSS.

Precise measurements of δ88/86Sr for twenty geological reference materials by double-spike MC-ICP-MS

Analytical method using double-spike and MC-ICP-MS can provide high efficiency of measurement for stable Sr isotopic compositions (δ88/86Sr). Here we report a method for high precision measurement of δ88/86Sr using MC-ICP-MS. A84Sr–87Sr double-spike was used to correct instrumental mass bias of stable Sr isotopes. Strontium was purified by a cation exchange resin (AG50W-X12, 200–400 mesh), and for samples with high Er, Yb, Lu, and Hf contents, an additional chromatographic purification procedure was applied. The potential effects of doubly charged ion isobaric inferences and different measurement conditions were rigorously evaluated to improve the analytical precision. Data quality was monitored by measuring three USGS reference materials (BHVO-2, AGV-2, and G-2) and two in-house reference materials, giving the long-term precision of 0.03‰ (2SD) for δ88/86Sr. Twenty reference materials, including nine igneous rocks, four sedimentary rocks, two sediments, and five soils, have δ88/86Sr values range from −0.06‰ to 0.35‰, showing resolvable isotope fractionation during magmatic and supergene processes. This study provides a useful standard dataset for inter-laboratory comparison of stable Sr isotope studies.

Multicentric Evaluation of SeeGene Allplex Real-Time PCR Assays Targeting 28 Bacterial, Microsporidal and Parasitic Nucleic Acid Sequences in Human Stool Samples.

Prior to the implementation of new diagnostic techniques, a thorough evaluation is mandatory in order to ensure diagnostic reliability. If positive samples are scarcely available, however, such evaluations can be difficult to perform. Here, we evaluated four SeeGene Allplex real-time PCR assays amplifying a total of 28 bacteria, microsporidal and parasitic nucleic acid sequence targets in human stool samples in a multicentric approach. In the assessments with strongly positive samples, sensitivity values ranging between 13% and 100% were recorded for bacteria, between 0% and 100% for protozoa and between 7% and 100% for helminths and microsporidia; for the weakly positive samples, the recorded sensitivity values for bacteria ranged from 0% to 100%; for protozoa, from 0% to 40%; and for helminths and microsporidia, from 0% to 53%. For bacteria, the recorded specificity was in the range between 87% and 100%, while a specificity of 100% was recorded for all assessed PCRs targeting parasites and microsporidia. The intra- and inter-assay variations were generally low. Specifically for some helminth species, the sensitivity could be drastically increased by applying manual nucleic acid extraction instead of the manufacturer-recommended automatic procedure, while such effects were less obvious for the bacteria and protozoa. In summary, the testing with the chosen positive control samples showed varying degrees of discordance between the evaluated Allplex assays and the applied in-house reference assays associated with higher cycle threshold values in the Allplex assays, suggesting that samples with very low pathogen densities might be missed. As the targeted species can occur as harmless colonizers in the gut of individuals in high-endemicity settings as well, future studies should aim at assessing the clinical relevance of the latter hint.

Multiplex agglutination-PCR (ADAP) autoantibody assays compared to radiobinding autoantibodies in type 1 diabetes and celiac disease

Multiplex Antibody-Detection by Agglutination-PCR (ADAP) assay was compared to singleplex standard radiobinding assays (RBA) to detect autoantibodies against insulin (IAA), GAD65 (GADA), islet antigen-2 (IA-2A), ZnT8 (ZnT8A) and tissue transglutaminase (TGA). Serum samples from 273 (114F/158M), 15–73 years of age healthy controls and 227 (109F/118M) newly diagnosed type 1 diabetes children, 1–11 years of age, were analyzed in both assay systems.The original WHO standard 97/550 and in-house reference standards for RBA were compared to ADAP. The ADAP and RBA generated parallel reference standards in all assays except TGA. Lower detection limits were observed in the ADAP assay for GADA,IAA and ZnT8A, markedly for TGA, but not for IA-2A. The Receiver Operating Characteristics (ROC) curve AUC analyses for pairwise comparison of ADAP with RBA showed no difference for GADA (n.s.), ADAP greater AUC for IAA (p = 0.005), RBA greater AUC for IA-2A (p = 0.0004) and ZnT8A (p < 0.0001) while ADAP TGA had a greater AUC compared to both RBA TGA-IgG (p < 0.0001) and TGA-IgA (p < 0.0001). These data suggest that the ADAP and RBA assays are comparable with equal performance for GADA, better ADAP performance for IAA while the RBA showed better performance in both IA-2A and ZnT8A associated with greater heterogeneity in autoantibody levels. The simultaneous analysis of 5 different autoantibodies by ADAP in sample volume reduced to only 4 μL and at an increased lower detection limit in all assays except IA-2A makes the ADAP automated autoantibody assay a distinct advantage for high throughput screening.