High Sample Volumes Research Articles

Optimizing porous asphalt mix design for permeability and air voids using response surface methodology and artificial neural networks

Easy infiltration of grouting material in Porous Asphalt Mixture (PAM) is one of the crucial phases in the construction of semi-flexible pavement (SFP). The gradation and compaction primarily influence the pores and their arrangement in PAM, which affects the air voids and permeability of porous mixes and effectiveness of slurry infiltration. Complexity of gradation and compaction makes it very difficult to predict air voids and permeability in PAM. Statistical analysis of these parameters based on experimental data requires very high volume of samples which is missing in literature. Compaction effort has been kept constant in most of them which otherwise is important for stability. Also, machine learning models to predict mix design of PAM has not been explored. This study investigates the PAM using systematic experimentation and modelling approaches such as Response Surface Methodology (RSM) and Artificial Neural Networks (ANN) to establish new and low trial-based guidelines for establishing a relationship between air voids and permeability greater than 0.12 cm/sec, while ensuring the stability of the mix. Using a Simplex Lattice design approach, five distinct types of aggregates were utilized as input variables and twenty-six PAM mixes were generated, which were subjected to 5 compaction levels. A total of 260 mixes were tested and then models were prepared based on RSM and ANN, which considered two output variables: air voids and permeability. RSM and ANN models demonstrated strong agreement between predicted values and actual experiment outcomes. It is observed that optimal permeability in PAM is achieved by adjusting aggregate proportions based on compaction levels. Proportion limits for low compaction suggested 60–70 % coarse aggregates and up to 20 % fines. For higher compaction (25–75 blows), it is recommended to increase the coarse aggregates up to70–90 % and limit fines up to 10 %. All proposed limits satisfied minimum permeability criteria (> 0.12 cm/sec) while ensuring the stability of the mixes. The proposed modelling approach offers greater efficiency and accuracy and reduces trial batch time, minimizing the need for destructive experiments and material wastage.

P-374 Preserving potential: evaluating transport media in ovarian tissue cryopreservation

Abstract Study question Which transport media best preserves ovarian tissue quality during overnight transport from the procurement site to the cryopreservation center for fertility preservation? Summary answer Buffered transport media, Oncofertility Consortium (OFC) holding medium, ORIGIO® Handling, or Custodiol® HTK preserved ovarian tissue better than non-buffered Ringer’s lactate. What is known already Ovarian tissue cryopreservation is the recommended mode of fertility preservation for prepubertal girls and is also used in certain adult patients. However, not every center offers cryopreservation services. Resulting in either costly and sometimes unfeasible patient travel or the overnight transportation of ovarian tissue to centralized processing center. Despite the high volume of samples processed and shipped to cryopreservation centers, current knowledge of the optimal transport media for ovarian tissues for fertility preservation is limited. Study design, size, duration To simulate a 24-hour shipping period, ovarian tissues from three adult organ donors were maintained for 24 hours at 4 °C in four distinct media: OFC holding media, Ringer’s Lactate, ORIGIO® Handling media, and Custodiol® HTK Solution. Samples were then cryopreserved and thawed. Tissue quality was assessed through histological evaluations and immunohistochemistry. Participants/materials, setting, methods To simulate a 24-hour shipping period, ovarian tissues from three adult organ donors were maintained for 24 hours at 4 °C in four distinct media: OFC holding media, Ringer’s Lactate, ORIGIO® Handling media, and Custodiol® HTK Solution. Samples were then cryopreserved and thawed. Tissue quality was assessed through histological evaluations and immunohistochemistry. Main results and the role of chance Eight conditions were compared to the control, which was analyzed upon receipt, including tissue following incubation in four distinct transport media with or without cryopreservation. The histological comparison of tissue following incubation in different transport media before cryopreservation revealed higher fibrosis in the tissue incubated in Ringer’s Lactate. All the tissue following cryopreservation had a higher fibrosis than the control. The immunohistochemical analysis did not demonstrate any significant differences in the mean percentage of apoptotic (Caspase-3+ and VASA+) oocytes from all (VASA+) oocytes among tissues incubated for 24 hours in different transport media but not cryopreserved. However, after cryopreservation, tissues incubated in Ringer’s Lactate had oocytes that were significantly more apoptotic (74.56%) than the control (P value = 0.0002); while tissues cryopreserved after incubation in OFC Holding Media (43.19%), Custodiol Solution (35.134%), and Origio Handling media (52.13%) were not different from the control (18.86%) group. Limitations, reasons for caution A significant limitation in our study is the absence of functional assessment of the tissues, limiting the insights into the complete physiological potential. Additionally, our study was conducted on adult donor tissue, which may not entirely reflect prepubertal ovarian characteristics. Wider implications of the findings These findings emphasize the crucial role of selecting the appropriate transport media for ovarian tissue cryopreservation, which could markedly impact fertility preservation outcomes. This research also sets the stage for standardized shipment protocols, enhancing the feasibility and success of ovarian tissue cryopreservation across centers and expanding access to fertility preservation. Trial registration number not applicable

Preconcentrating extractive polymeric network in paper-based sensing of copper

The concept of using an extractive polymeric sensing network on a paper-based substrate for the pretreatment of samples in the colorimetric analysis of heavy metals is demonstrated with the determination of Cu2+ in aqueous samples. The newly developed paper-based device combines the advantages of both liquid–liquid extraction-based separation and the analytical capabilities of microfluidic paper-based analytical devices (μPADs). The preconcentrating extractive polymeric network composed of 53.5 wt% poly(vinyl chloride) (PVC), 45 wt% extractant (di-(2-ethylhexyl)phosphoric acid (D2EHPA)) and 1.5 wt% dye (1-(2′-pyridylazo)-2-naphthol (PAN)), coated on filter paper, shows good water-wicking properties, allowing the transmission of high sample volumes. This results in significant analyte preconcentration. The extractant (D2EHPA) selectively binds Fe3+ and Zn2+ which otherwise will compete with Cu2+ in its colour reaction with PAN at the chosen pH of 3.0. The device’s capacity to process higher sample volumes resulting in higher sensitivity is increased by the integration of several filter paper absorption layers. The optimized device is characterized by a limit of detection (LOD) for tap and environmental waters of 3.4 and 9.6 µg/L, respectively; stability of 51 days if stored in a refrigerator; and total analysis time (from sample insertion to colour detection) of less than 5 min.

Comparative assessment of Nanotrap and polyethylene glycol-based virus concentration in wastewater samples.

Wastewater-based epidemiology is now widely used in many countries for the routine monitoring of SARS-CoV-2 and other viruses at a community level. However, efficient sample processing technologies are still under investigation. In this study, we compared the performance of the novel Nanotrap® Microbiome Particles (NMP) concentration method to the commonly used polyethylene glycol (PEG) precipitation method for concentrating viruses from wastewater and their subsequent quantification and sequencing. For this, we first spiked wastewater with SARS-CoV-2, influenza and measles viruses and norovirus and found that the NMP method recovered 0.4%-21% of them depending on virus type, providing consistent and reproducible results. Using the NMP and PEG methods, we monitored SARS-CoV-2, influenza A and B viruses, RSV, enteroviruses and norovirus GI and GII and crAssphage in wastewater using quantitative PCR (qPCR)-based methods and next-generation sequencing. Good viral recoveries were observed for highly abundant viruses using both methods; however, PEG precipitation was more successful in the recovery of low-abundance viruses present in wastewater. Furthermore, samples processed with PEG precipitation were more successfully sequenced for SARS-CoV-2 than those processed with the NMP method. Virus recoveries were enhanced by high sample volumes when PEG precipitation was applied. Overall, our results suggest that the NMP concentration method is a rapid and easy virus concentration method for viral targets that are abundant in wastewater, whereas PEG precipitation may be more suited to the recovery and analysis of low-abundance viruses and for next generation sequencing.

A sensorised, microfluidic Ussing chamber manufactured by stereolithography and soft lithography techniques

Motivated by the need for improved platforms in biomedical research, this study addresses challenges associated with traditional Ussing chamber systems, widely used in studying biological barriers like the epithelial barrier of the gut. These challenges include complexity, high sample volumes, and limited compatibility. By combining stereolithography and soft lithography techniques, a microfluidic Ussing chamber is manufactured, overcoming these limitations, and incorporating compatibility with microscopy. Validation through Trans-Epithelial Electrical Resistance (TEER) measurements confirms its efficacy in assessing ion permeability dynamics, utilizing Caco-2 cell monolayers. The study showcases the capability of the manufactured chamber for sensing impact of calcium on tight junctions.

Simple performance of the planar SOS-Umu-C–FLD genotoxicity bioassay shown for perfume and packaging material analysis

Planar bioassays are powerful, sustainable tools for nontargeted detection of hazardous compounds in complex samples. They provide more information on a sample than corresponding in vitro assays and are more sustainable in terms of plastic material and solvent consumption. However, instrument investment costs for high-performance thin-layer chromatography are high. Hence, the question arose of whether the sophisticated sensitive planar genotoxicity bioassay could be performed manually without instrumentation under simple conditions. Cheaper plate types were studied as well as manual application of the samples, cell suspension, and substrate solution. As a result, genotoxic compound zones were detected as rose-colored or orange fluorescent resorufin end-product formed upon contact of the genotoxins in tested perfume and packaging materials with a genetically modified Salmonella Typhimurium strain. The simple performance was found to be possible for low sample application volumes. Knowledge on neutral pH value and thickness of the adsorbent layer were further key aspects. Manual spraying was found to be superior to manual immersion if excess liquid was avoided. For high sample volumes and a higher level of standardization, the open-source 2LabsToGo system was proposed as excellent option for low investment costs. Its very low instrumental footprint and the straightforward prioritization strategy help analytical chemistry to balance between technology and nature/ecology to reduce the instrumental footprint and planetary overshoot.

Molecular analysis of nasal secretion to identify Alzheimer’s disease

AbstractBackgroundIncreasing clinical evidence shows significant drainage of cerebrospinal fluid (CSF) and its metabolites along the olfactory route into the nose. Nasal secretion‐based analysis of key Alzheimer biomarkers was compared to CSF‐based analysis in different cohorts.MethodWe conducted a multicenter, prospective cohort study involving patients with cognitive impairment and availability of CSF measurement of the 4 AD‐specific proteins. Cohorts were defined based on CSF biomarker classification following NIA‐AA ATN research diagnostic framework and clinical findings. Nasal secretion (NS) was collected from the vicinity of the olfactory cleft. [NosetestAD] was performed for subsequent in vitro analysis.ResultFor this analysis, 39 patients with cognitive impairment were included at 3 university clinics in Germany. The nasal sampling procedure [noseabsorb] is painless (VAS 2 ±1.7) and yields a high sample volume (mean 450 μl). [NosetestAD] allows for precise quantification in size and relative protein amount of AD biomarkers pTau181, total Tau, Amyloid‐ß1‐40 and Amyloid‐ß1‐42. By using an automated protein separation and immunodetection system, we are able to generate a protein specific biomarker signature for all 4 AD‐associated proteins (pTau, tTau, Aß42,Aß40) characterizing parameters such as oligomerization state and relative protein amount. Further, a highly significant discrimination of amyloid pathology (A+ vs. A‐) was achieved with an AUC of 0.94 and p‐value <0.001.ConclusionNasal secretion constitutes a safe, minimally invasive access to CSF. Therefore, it is highly suitable for detection and monitoring of AD relevant biomarkers. Within a cohort of patients with cognitive impairment A+ patients can be distinguished from A‐ patients based on their diseases state specific individual protein‐specific biomarker signatures and the different levels of oligomeric or monomeric structures. Thus, our proprietary diagnostic platform, detects and assesses a broad panel of brain derived biomarkers simultaneously and allows for intraindividual monitoring and possibly disease staging in nasal secretion.

Microfluidic pumps for cell sorting.

Microfluidic cell sorting has shown promising advantages over traditional bulky cell sorting equipment and has demonstrated wide-reaching applications in biological research and medical diagnostics. The most important characteristics of a microfluidic cell sorter are its throughput, ease of use, and integration of peripheral equipment onto the chip itself. In this review, we discuss the six most common methods for pumping fluid samples in microfluidic cell sorting devices, present their advantages and drawbacks, and discuss notable examples of their use. Syringe pumps are the most commonly used method for fluid actuation in microfluidic devices because they are easily accessible but they are typically too bulky for portable applications, and they may produce unfavorable flow characteristics. Peristaltic pumps, both on- and off-chip, can produce reversible flow but they suffer from pulsatile flow characteristics, which may not be preferable in many scenarios. Gravity-driven pumping, and similarly hydrostatic pumping, require no energy input but generally produce low throughputs. Centrifugal flow is used to sort cells on the basis of size or density but requires a large external rotor to produce centrifugal force. Electroosmotic pumping is appealing because of its compact size but the high voltages required for fluid flow may be incompatible with live cells. Emerging methods with potential for applications in cell sorting are also discussed. In the future, microfluidic cell sorting methods will trend toward highly integrated systems with high throughputs and low sample volume requirements.

Simultaneous estimation of five biomarkers of neuroprotective herb Ashwagandha NMITLI-118R AF1 in rat plasma and brain using LC-ESI-MS/MS: Application to its pharmacokinetic and stability studies

Withania Somnifera (WS) is a popular nutritional supplement in the USA, Europe, and Asia, known for its pharmacological effects on neurological disorders. However, the bioanalytical method development, validation, and pharmacokinetics of WS NMITLI-118R AF1 biomarkers Withanolide A (WLD A), Withanone (WNONE), Withanolide B (WLD B), Withaferin A (WF A), and 12 Deoxywithastramonolide (12 DEOXY) in rats have not been comprehensively explored. This study aimed to develop and validate a sensitive and selective LC-ESI-MS/MS method for these biomarkers in male Sprague Dawley rats plasma and brain matrix. Rats were divided into eight groups, each containing five rats. A plant extract of NMITLI-118R AF1 at 50 mg/kg was orally administered to the rats for in-vivo pharmacokinetic investigation. All the analytes had a linear calibration curve (r2 > 0.999), and intra-day and inter-day precision (%) were found in the range of 2.46 – 13.71% and accuracy were within the acceptable range (±15%). The biomarkers of NMITLI-118R AF1 were found stable in in-vitro plasma and simulated gastro-intestinal fluids. The observed (Cmax) and (Tmax) values for the biomarkers in the systemic circulation were WLD A (5.59 ± 0.34 ng/mL, Tmax 1.00 ± 0.00 h), WNONE (6.28 ± 0.41 ng/mL, Tmax 0.95 ± 0.11 h), WLD B (6.45 ± 2.87 ng/mL, Tmax 0.95 ± 0.11 h), WF A (6.50 ± 0.27 ng/mL, Tmax 1.00 ± 0.00 h), and 12 DEOXY (5.68 ± 0.39 ng/mL, Tmax 1.00 ± 0.00 h). In contrast to the old method, our approach exhibits a lower limit of quantification (LLOQ), shorter run time (less than10 min), and enables the detection of WF A and WNONE in fresh rat plasma by other quantitative analysis of mass spectrometry (m/z) [M]+. Shows high sample volumes for both, larger plasma volumes, costlier sample collection techniques dried blood spot (DBS), more expensive solid phase extraction techniques (SPE) and longer analysis time 14 min. Moreover, our method requires a smaller sample volume 10 µL, offers faster analysis time 4 min, and achieves a higher sensitivity 1 ng/mL. This is the first report of a comprehensive study on in-vitro and in-vivo pharmacokinetics of NMITLI-118R AF1 biomarkers, which may aid in further pre-clinical and clinical trial investigations.

Scatter gram patterns of hematological malignancies in Sysmex XN series

Background: Scatter grams-graphic depictions of the cell populations in a blood sample are produced by automated analyzers. These scatter grams can be employed to screen for various hematological conditions, including anemia, leukemia, and other blood disorders by examining the scatter gram’s cell population distribution and patterning. Aims and Objectives: In this study, we wish to study the scatter grams generated by the Sysmex XN-series analyzer for confirmed cases of hematological malignancies. Materials and Methods: In this study, 58 cases of hematologic malignancies diagnosed based on peripheral smear findings and bone marrow morphology data were extracted. We retrieved the complete blood count and scatter gram patterns analyzed by Sysmex XN-1000 analyzer for these cases along with peripheral smear findings. Scatter gram patterns in white blood cell differential and white cell nucleated were analyzed correlating with peripheral smears. Results: Among 58 cases, 20 cases were acute myeloid leukemia (6 were acute promyelocytic leukemia), 32 cases were chronic myeloid leukemia, and 5 cases were chronic lymphocytic leukemia. Unique scatter gram patterns were seen with different leukemias. Conclusion: Pattern analysis of scatter grams has the potential to improve the efficiency and accuracy of leukemia diagnosis and also help to prioritize the evaluation of cases that require further testing, such as molecular and cytogenetic evaluations, especially in limited resources or high sample volumes conditions.

Optimal fluid volume for detecting malignancy in serous effusions: a single institution experience

Detection of malignant cells in serous fluids is an indicator of advanced stage of malignancy and is critical in clinical management decisions and prompt treatment initiation. The minimum volume which is ideal for detecting malignancy in serous fluid is not well established. In this study, we aim to identify optimal volume that will be ideal for adequate cytopathological diagnosis. A total of 1597 samples of serous fluids from 1134 patients were included in the study. Samples were diagnosed based on International System for Reporting Serous Fluid Cytopathology (ISRSFC). Clinicopathologic results from different diagnostic groups were compared and statistically analyzed. Pleural fluids comprised 890 (55.7%) specimens, followed by 456 (28.6%) peritoneal, 128 (8%) ascites, and 123 (7.7%) pericardial fluid specimens. The majority were negative for malignancy (1138, 71.3%), followed by malignant (376, 23.5%), atypical (59, 3.7%), and suspicious for malignancy (24, 1.5%). Malignancy was identified in sample with volumes from 5 mL to 5000 mL. Rate of detection of malignant cells increased significantly with higher sample volumes. For malignancy detection the optimal volume for overall serous fluid is 70 mL. Pericardial fluid is an exception, with lower mean volume and significantly lower proportion of cases with malignant diagnosis. Our study indicates that higher fluid volumes have a higher rate of malignancy detection and a low false-negative rate. We recommend a minimum of 70 mL of serous fluid for optimal cytopathologic examination and malignancy detection. Pericardial fluid is an exception, with lower mean volume and thus lower requirement.

Efficient Purification of Polyhistidine-Tagged Recombinant Proteins Using Functionalized Corundum Particles.

Immobilized metal affinity chromatography (IMAC) is a popular and valuable method for the affinity purification of polyhistidine-tagged recombinant proteins. However, it often shows practical limitations, which might require cumbersome optimizations, additional polishing, and enrichment steps. Here, we present functionalized corundum particles for the efficient, economical, and fast purification of recombinant proteins in a column-free format. The corundum surface is first derivatized with the amino silane APTES, then EDTA dianhydride, and subsequently loaded with nickel ions. The Kaiser test, well known in solid-phase peptide synthesis, was used to monitor amino silanization and the reaction with EDTA dianhydride. In addition, ICP-MS was performed to quantify the metal-binding capacity. His-tagged protein A/G (PAG), mixed with bovine serum albumin (BSA), was used as a test system. The PAG binding capacity was around 3 mg protein per gram of corundum or 2.4 mg per 1 mL of corundum suspension. Cytoplasm obtained from different E. coli strains was examined as examples of a complex matrix. The imidazole concentration was varied in the loading and washing buffers. As expected, higher imidazole concentrations during loading are usually beneficial when higher purities are desired. Even when higher sample volumes, such as one liter, were used, recombinant protein down to a concentration of 1 µg/mL could be isolated selectively. Comparing the corundum material with standard Ni-NTA agarose beads indicated higher purities of proteins isolated using corundum. His6-MBP-mSA2, a fusion protein consisting of monomeric streptavidin and maltose-binding protein in the cytoplasm of E. coli, was purified successfully. To show that this method is also suitable for mammalian cell culture supernatants, purification of the SARS-CoV-2-S-RBD-His8 expressed in human Expi293F cells was performed. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents for 1 g of functionalized support or 10 cents per milligram of isolated protein. Another advantage of the novel system is the corundum particles' extremely high physical and chemical stability. The new material should be applicable in small laboratories and large-scale industrial applications. In summary, we could show that this new material is an efficient, robust, and cost-effective purification platform for the purification of His-tagged proteins, even in challenging, complex matrices and large sample volumes of low product concentration.

Expanding accessible 3D sample size in lab-based X-ray nanotomography without compromising resolution

State-of-the-art lab-based X-ray nanotomography enables versatile and well-accessible three-dimensional imaging of samples with spatial resolutions comparable to synchrotron setups. By its nature, the technique brings a trade-off between high spatial resolution and accessible sample volume. A so-called ‘stitching approach’ can overcome this challenge by extending the accessible three-dimensional volume without compromising spatial resolution and is demonstrated in a state-of-the-art lab-based X-ray microscope on two porous, low-Z material systems: macroporous zeolite particles and supported catalytically active metal solutions. Fourier ring and shell correlation are utilized to calculate the achievable two- and three-dimensional resolution of the X-ray microscope and the tomographic reconstructions, respectively. The excellent performance of the stitching approach is evaluated by comparing three-dimensional reconstructions of identical sample volumes obtained with and without stitching at different magnifications. The influence of illumination time, depth of field and number of projections are investigated by comparing the spatial resolution of the experiments with theoretical resolution limits.

In vitro evaluation of immunomodulatory activities of goat milk Extracellular Vesicles (mEVs) in a model of gut inflammation

Gut represents a major immunological defense barrier with mucosal immune system and intestinal epithelial cells (IECs). In all intestinal diseases, in particular inflammatory bowel disease (IBD), both the absorption and the local immune system are compromised and alternative effective therapies are sought after. Extracellular Vesicles (EVs) have the capability to regulate immune cells within the inflammatory microenvironment, by dampening inflammation and restoring intestinal barrier integrity. Recently, the immune-modulatory role of EVs has also been confirmed for milk EVs (mEVs), notable for their easy production, high sample volumes, cost-effective scalable production and non-toxic and non-immunogenic behavior. In this context, the aim of this study was to evaluate goat mEV anti-inflammatory and immuno-modulating effects on an in vitro model (IPEC-J2) of intestinal inflammation through gene expression evaluation with RT-qPCR and cytokine release dosage with ELISA test. After the establishment of a pro-inflammatory environment due to LPS stimuli, IL6, CXCL8, IL12p35, IL12p40, IFNB, IL18, TLR7 and NOS2 resulted significantly up-regulated in stimulated IPEC-J2 cells compared to those of the basal culture. After 48 h of mEV treatment in inflamed IPEC-J2 a partial restoration of initial conditions was detected, with the IL18 and IL12p40 significant down-regulation, and IL12p35, EBI3, TLR7, BD1 and BD3 up-regulation. IL-18 reduced protein production was also detected in supernatants. Moreover, a decrease of MMP9 and NOS2 together with a strong up-regulation of MUC2 indicated a recovery of cellular homeostasis and, therefore, potential beneficial effects on the intestinal mucosa. Nevertheless, 48 h post-treatment, an increased gene expression and protein release of IL-8 was observed. This paper is one of the firsts to assess the effect of goat mEVs and the first one, in particular, of doing this on an in vitro model of gut inflammation. The obtained results show a potential capability of goat mEVs to modulate inflammation and to play beneficial effects on the intestinal mucosa.

Vortex-assisted magnetic dispersive solid phase extraction using Tanacetum extract followed by dispersive liquid–liquid microextraction for the extraction and preconcentration of Co(II) and Ni(II) from high volume of water samples

Vortex-assisted magnetic dispersive solid phase extraction using Tanacetum extract followed by dispersive liquid–liquid microextraction for the extraction and preconcentration of Co(II) and Ni(II) from high volume of water samples

Abstract 5156: Multiplexed EGFR mutation detection with C797S and T790M allelic context using six-color digital PCR

Abstract Non-small cell lung cancer (NSCLC) patients harboring activating mutations in epidermal growth factor receptor (EGFR) are benefited from Tyrosine Kinase Inhibitor (TKI) targeted therapy. However, more than 50% of patients receiving the first or second generations of TKI develop a resistance mutation, EGFR-T790M, and they will need to switch to the third-generation TKI (Osimertinib). Upon treatment, a second acquired mutation, EGFR-C797S, leads to the resistance to Osimertinib. If the C797S and T790M mutations are located in trans, a combination of the first and third-generation of TKI(s) may offer some benefit to the patient. The impact of the allelic context to the subsequent treatment is yet to be established and thus an assay identifying C797S and T790M allelic context may serve as a useful research tool. In recent years, digital PCR has emerged to be an ultra-sensitive method in non-invasive identification and monitoring of cancer mutations. However, compared to qPCR and NGS methods, current digital PCR assays are limited by the number of biomarkers that can be incorporated in a single assay and the maximum sample volume that can be screened in one test. In this study, we describe a multiplex digital PCR assay for quantitative detection of EGFR sensitizing and resistance mutations, and identification of C797S and T790M allelic context, using a 6-color digital PCR system that allows high sample volume utilization. The sensitivity and specificity of this multiplex dPCR assay were tested using a panel of contrived cell-free DNA samples. Individual mutations can be detected at as low as 0.2% fractional abundance level. Furthermore, we compared the performance of the multiplex with the 2-color droplet digital PCR Expert Design Assays on the Bio-Rad QX200 ddPCR system, using a commercial multiplex cfDNA reference standard set. The multiplexed EGFR mutation assay on the 6-color Digital LightCycler® System is a promising method for rapid and sensitive monitoring of EGFR mutations in cell-free DNA. Citation Format: Wei Yang, Mari Christensen, Jennifer K. Chan, Julie Tsai, Nancy Patten, Ha B. Tran, Grant R. Hillman, Yu Chuan Tai, Patrick Bogard, Claudia M. Litterst, Victoria H. Brophy, Nick Newton, Christopher D. Nelson, Wouter J. Pattje. Multiplexed EGFR mutation detection with C797S and T790M allelic context using six-color digital PCR [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 5156.

Preparation of a magnetic sorbent based on Tanacetum extract and its application in the extraction of Cu(II) and Pb(II) ions from milk performed in a narrow-bore tube followed by dispersive liquid–liquid microextraction

In this work, an efficient magnetic sorbent has been prepared using the extract of Tanacetum leaves and used in magnetic dispersive solid phase extraction of Cu(II) and Pb(II) ions from high volume of milk samples performed in a narrow-bore tube. For this purpose, first, the sorbent was prepared by mixing Fe3O4 magnetic nanoparticles and Tanacetum leaves extract. After precipitation of the milk proteins with trichloroacetic acid, the sedimented compounds were removed and the upper solution was filled into the narrow-bore tube. Then after that, the mg-level of the prepared sorbent was added. After doing the extraction procedure, the sorbent particles were removed from the tube in the presence of a magnetic field. The adsorbed analytes were desorbed by methanol and to achieve low detection limits, the eluent obtained from the first step was subjected to dispersive liquid-liquid microextraction. Finally, the enriched analytes were monitored by flame atomic absorption spectrometry. The important parameters affecting the method efficiency such as the sorbent amount, type and volume of elution and extraction solvents, vortex time, pH, and ionic strength were studied. Under optimized conditions, the proposed method showed linear ranges of 0.50–40 and 0.75–40 µg L−1 for Cu(II) and Pb(II) ions, respectively. Limits of detection were obtained 0.16 and 0.22 µg L−1 for Cu(II) and Pb(II), respectively. Enrichment factors of 182.5 and 173.7 and extraction recoveries of 95.8 % and 91.2 % were obtained for Cu(II) and Pb(II), respectively. The relative standard deviations (intra- and inter-day precisions) for 5 and 25 µg L−1 of each cation were between 2.4 % and 4.3 %.

Reverse transcription PCR to detect low density malaria infections

Background: Targeted malaria elimination strategies require highly sensitive tests to detect low density malaria infections (LDMI). Commonly used methods for malaria diagnosis such as light microscopy and antigen-based rapid diagnostic tests (RDTs) are not sensitive enough for reliable identification of infections with parasitaemia below 200 parasites per milliliter of blood. While targeted malaria elimination efforts on the Thailand-Myanmar border have successfully used high sample volume ultrasensitive quantitative PCR (uPCR) to determine malaria prevalence, the necessity for venous collection and processing of large quantities of patient blood limits the widespread tractability of this method. Methods: Here we evaluated a real-time reverse transcription PCR (RT-qPCR) method that reduces the required sample volume compared to uPCR. To do this, 304 samples collected from an active case detection program in Kayin state, Myanmar were compared using uPCR and RT-qPCR. Results: Plasmodium spp. RT-qPCRconfirmed 18 of 21 uPCR Plasmodium falciparum positives, while P. falciparum specificRT-qPCR confirmed 17 of the 21 uPCR P. falciparum positives. Combining both RT-qPCR results increased the sensitivity to 100% and specificity was 95.1%. Conclusion: Malaria detection in areas of low transmission and LDMI can benefit from the increased sensitivity of ribosomal RNA detection by RT-PCR, especially where sample volume is limited. Isolation of high quality RNA also allows for downstream analysis of malaria transcripts.

An Alternative Strategy for Screening and Confirmation of 330 Pesticides in Ground- and Surface Water Using Liquid Chromatography Tandem Mass Spectrometry.

The presence of pesticide residues in water is a huge worldwide concern. In this paper we described the development and validation of a new liquid chromatography tandem mass spectrometric (LC-MS/MS) method for both screening and quantification of pesticides in water samples. In the sample preparation stage, the samples were buffered to pH 7.0 and pre-concentrated on polymeric-based cartridges via solid-phase extraction (SPE). Highly sensitive detection was carried out with mobile phases containing only 5 mM ammonium formate (pH of 6.8) as an eluent additive and using only positive ionization mode in MS/MS instrument. Hence, only 200-fold sample enrichment was required to set a screening detection limit (SDL) and reporting limit (RL) of 10 ng/L. The confirmatory method was validated at 10 and 100 ng/L spiking levels. The apparent recoveries obtained from the matrix-matched calibration (5–500 ng/L) were within the acceptable range (60–120%), also the precision (relative standard deviation, RSD) was not higher than 20%. During the development, 480 pesticides were tested and 330 compounds fulfilled the requirements of validation. The method was successfully applied to proficiency test samples to evaluate its accuracy. Moreover, the method robustness test was carried out using higher sample volume (500 mL) followed by automated SPE enrichment. Finally, the method was used to analyze 20 real samples, in which some compounds were detected around 10 ng/L, but never exceeded the assay maximum level.

Monitoring soil water content and its salinity with high-precision and low-cost in-situ sensors

The article presents an in situ sensor to monitor soil moisture and water salinity by measuring soil permittivity and conductivity at a frequency 24 MHz via an admittance approach. It addresses difficulties encountered in other permittivity-based sensors. A self-balanced bridge provides on one measurement a digital output linear to capacitance and conductance of electrodes in soil with low offset, high phase resolution, and taking into account all parasitic impedances. Circuit design and procedures to achieve it are described, using electric circuit theory and component specifications. Probe geometry, two parallel cylinders, permits insertion in soil from surface, low disturbances and a high sample volume around electrodes. It provides an analytical relation to convert admittance to soil permittivity. Calibration of a sensor with air, alcohols and water fixes its sensitivity and residual phase and inductance for an uncertainty lower than 2% over a range up to 80 and 200 mS m–1. Identical sensors can use same parameters after adjusting in one simple operation bridge phases. Thermal drift and its consequences on soil variable measurement are investigated and physical origins identified. It permits to reduce the phase drift within ±150 ppm °C– 1 rad to keep accuracy.