Thermo ScientificTM Research Articles

Fast Determination of Propofol in Human Plasma Using C18-Functionalized Magnetic Nanomaterials Followed by Supercritical Fluid Chromatography.

Novel C18-functionalized magnetic nanomaterials; i.e., C18@poly-styrene-divinylbenzene-glycidyl methacrylate-Fe3O4 (C18@PS-DVB-GMA-Fe3O4) have been synthesized by using N, N-dimethyloctadecylamine as modifying agent, which could be beneficial to remove the blood phospholipids. The C18@PS-DVB-GMA-Fe3O4 nanoparticles have been used and evaluated in the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) procedure for human plasma prior to the analysis of propofol by supercritical fluid chromatography (SFC). In the QuEChERS procedure, human plasma samples are directly mixed with extraction solvent and C18@PS-DVB-GMA-Fe3O4 nanoparticles, and the extraction and cleanup procedures have been accomplished simultaneously. The SFC separation was performed with a C18 column (Thermo Scientific™ Acclaim™ 120, 250 × 4. 6mm, 5μm) within 5min, using thymol as the internal standard. Supercritical carbon dioxide was used as the mobile phase with methanol as the cosolvent at the flow rate of 1.0mL/min. The column temperature was 38°C, and detection wavelength was 275nm. A good linearity was obtained among the propofol concentration range of 0.5-10mg/L (R2 = 0.9997) with the limit of detection of 0.17mg/L. Recoveries were in the range of 76.5-91.9%, with RSD less than 7.9%. These results suggested that method is convenient, rapid with high accuracy and little matrix effect, and suitable for rapid determination of propofol plasma concentration.

Optimising Nanoparticle Dispersion Time for Enhanced Thermomechanical Properties in DGEBA-Based Shape Memory Polymer Composites

Shape-memory polymers (SMPs) are smart materials that can change shape upon an external stimulus. This phenomenon is called the shape memory effect (SME), which is caused by entropy change due to rapid molecular motion in the polymer segments. Due to the inherently weak thermomechanical properties, use of SMPs is limited in many engineering applications. Therefore, SMPs are often reinforced with fibres and nanoparticles (NPs). NPs offered greater flexibility due to their superior physical, chemical, electrical, mechanical, and thermal properties. However, the homogeneous distribution of NPs is crucial for composition’s stability and enhancement of the base material’s properties. Among the different techniques used for dispersing NPs, ultrasonic irradiation has shown excellent emulsifying and crushing performance. The sonication process is essential for mitigating agglomerates; however, prolonged sonication time probably increases epoxy temperature, micro-bubbles, cavitation, breaking apart molecules and finally degrading the epoxy resin performances. This paper provides critical insight of nanoparticle dispersion into diglycidyl ether of bisphenol A epoxies (DGEBA). DGEBA epoxy resin was added to TiO2 NPs and sonicated for 60 min with 5 min intervals while the temperature of epoxy was maintained below 60oC by using a water cooling throughout the sonication process. The process parameters such as amplitude, mode, epoxy volume and the weight percentage of NPs were kept constant. After each sonication step, Fourier-transform infrared spectroscopy (FTIR) was performed using Thermo Scientific™ and analysed through OMNIC™ Professional quantitation software. In accordance with FTIR results, until 30 min of the sonication, DGEBA resin was not degraded. In order to confirm the performances and the reinforcing effect of NPs, thermo-mechanical and shape memory properties were compared with the neat specimen. The outcomes of this research have suggested quick guidance to find optimum NP dispersion time for DGEBA resins, which has been hardly studied before.

Comparison of two commercial broth microdilution panels for multidrug-resistant Gram-negative bacteria: Thermo Scientific™ Sensititre DKMGN vs. Beckman Coulter MicroScan NMDRM1.

Antimicrobial susceptibility testing (AST) using broth microdilution (BMD) is usually the reference method to obtain accurate minimum inhibitory concentrations and optimally manage infections with resistant organisms. Several commercial dry BMD are available for AST in clinical laboratories. Two commercial BMD panels for testing of multidrug-resistant Gram-negative bacteria were compared: the Thermo Scientific™ Sensititre DKMGN and the Beckman Coulter NMDRM1, for 17 antimicrobial agents. A total of 207 isolates were tested: three ATCC strains and one NCTC strain, six quality control strains from the Belgian National Antimicrobial Committee, and 197 clinical isolates, including carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2023 breakpoints version 13.1 were used to assign susceptibility categories. Overall, the categorical agreement (CA) and essential agreement (EA) were both above 90%, but several useful antibiotics for the treatment of multi-resistant organisms showed CA and EA under 90%, that is, meropenem, imipenem, and colistin for Enterobacterales and meropenem and colistin for P. aeruginosa. For Enterobacterales, the NMDRM1 panel showed a significantly higher resistance rate for meropenem, imipenem, amikacin, and colistin. For carbapenems, the minimal inhibitory concentrations (MICs) were underestimated by the DKMGN panel, as already pointed out by a warning on the EUCAST website. To better assess carbapenem susceptibility in carbapenem-resistant organisms, the DKMGN panel now requires the use of a higher inoculum in the insert kit. However, for a given isolate whose susceptibility to carbapenems is not known, there is a risk of underestimating the MIC values. Our results show that colistin testing remains a challenge, highlighting the urgent need for the development of more accurate commercial methods. The use of a single commercial method cannot guarantee good precision in the determination of the MIC value for colistin.

A-043 A Fast, Novel and Analytically Sensitive Clinical Research Method for the Analysis of Free Testosterone using UPLC-MS/MS

Abstract Background The aim of this work was to develop an analytically sensitive, rapid clinical research method for the analysis of free testosterone in serum using a small sample volume. Laboratories have found this difficult, partly due to the inherent low concentrations (less than 3% of total testosterone is free), technical challenges and variability in results have been noted with equilibrium dialysis, which is considered the gold standard. Methods Testosterone certified reference material (Merck, UK) was used to create calibrators in 52.75 mM HEPES buffer adjusted to pH 7.4 to mimic the ionic environment of serum. Precision was evaluated using in-house QC materials prepared in serum (Golden West Biologicals, USA and BioIVT, UK). 200 µL serum was incubated at 37°C with 400 µL pH-adjusted 52.75 mM HEPES buffer for 2 hours, mixing at 800 r.p.m. with Rapid Equilibrium Analysis (RED) inserts and plates (Thermo Scientific™, UK). Testosterone-13C3 (Merck, UK) was spiked into resultant diasylate, then liquid-liquid extraction (LLE) was used to concentrate and clean the sample. The ACQUITY™ UPLC™ I-Class System with the Waters™ ACQUITY UPLC BEH C18, 1.7µm, 2.1 x 100 mm Column using a water/methanol/ammonium fluoride gradient and the Waters Xevo™ TQ Absolute Mass Spectrometer were used for quantification. An accelerated protocol for equilibrium dialysis, using a 2-hour incubation and fast mixing step was used (4 hours or longer is more typical historically) though it is essential to control the pH of the dialysis buffer and, particularly, the temperature of the chamber. Results No significant carryover or matrix effects were demonstrated with linearity from 0.5-650 pg/mL. Addition of high concentrations of endogenous substances (albumin, bilirubin, creatinine, cholesterol, triglycerides and uric acid) did not affect quantification. Analytical sensitivity investigations indicated the analytical sensitivity would allow precise quantification (≤20%CV and ≤15% bias) at 0.50 pg/mL. Coefficients of variation (CV) for total precision and repeatability on 5 analytical runs for low, mid and high QCs at approximately 2.79, 8.80 and 134 pg/mL respectively were all ≤ 8.4% (n=25). Accuracy was assessed by analyzing 45 male external quality assurance samples (NEQAS, UK) ranging in concentration from 50.1-268.6 pg/mL and the Bland-Altman agreement showed a small negative bias of -7.6% compared with the all-laboratory trimmed mean (ALTM), though excellent agreement overall. When LC-MS/MS determined concentrations were processed against Vermeulen equation determined concentrations (total testosterone and SHBG concentrations were provided by the scheme, a concentration of 45 g/L albumin was assumed), again good agreement was shown by the Bland-Altman bias of -7.7%. Conclusions A novel clinical research method using a fast equilibrium dialysis procedure with LLE and LC-MS/MS analysis with only 200 µL of serum has been demonstrated for clinical research use. Sub pg/mL analytical sensitivity, excellent linearity and precision, with minimal matrix effects and a strong agreement with an EQA scheme were established.

B-018 Design and Development of MASTM Diabetes Max Controls, Ready-to-Use Plastic Tube Format

Abstract Background The Thermo Scientific™ MAS™ Controls are assayed controls to monitor assay performance within clinical laboratory settings. The user can compare observed results of controls with their expected ranges as a means of assuring consistent performance of both reagent and instrument. The objective of developing MAS™ Ready-to-Use Tube Controls is to provide MAS™ controls in a new automation-friendly plastic tube format, as an alternative to the current glass vials. Diabetes Max Control is the first MAS™ product designed and developed in this ready-to-use format to be placed directly on the analyzer. The new format will increase efficiency and allow for on-board refrigerated storage, and it is expected to reduce material loss and contamination. It contains Hemoglobin A1c formulated in whole blood matrix to mimic patient specimens. In this presentation we summarize the Feasibility, Development, Verification, and Validation results of MAS™ Diabetes Max Controls. Methods The performance was assessed by conducting the following studies for MAS™ Diabetes Max Tube Controls. The analyte Hemoglobin A1c was measured in functional studies to evaluate and verify the product performance. Other studies were also designed and performed to assess the product usability. Results MAS™ Diabetes Max Control results demonstrated comparable performance and Fit/Form/Function criterion on specified platforms, particularly the TOSOH™ platform. All feasibility studies provided very promising results passing per protocol criterion. Design Verification and Validation studies ensured product integrity and passed specifications for proposed claims. Process Validation is in progress with data being collected for both Value Assignments and Real Time Stability. Conclusions MAS™ Diabetes Max Controls provided in ready-to-use tubes met the design specification criteria. We believe that MAS™ Diabetes Max Controls will contribute to the increased efficiency of core lab workflow by allowing the analyzer(s) to aspirate controls directly from the tube and allow for control storage in an on-board refrigeration unit.

B-248 Enhanced Detection of of Hemoglobin Variants in Clinical Research using Dried Blood Spot and HRAM MS

Abstract Background Hemoglobinopathies are one of the most prevalent genetic disorders, with more than 330,000 affected births occurring annually. Common techniques used in Hb analysis are electrophoretic and chromatographic assays. However, there are challenges to differentiate isomers (i.e., between Hb C and Hb E), very similar masses, or new variants due to their lack of resolution. Mass spectrometry (MS) gained popularity in clinical research because of its robustness and versatility in analyzing a wide range of samples and analytes from small molecules to proteins. Here, we present the top-down analysis for enhanced detection of various hemoglobin variants using Orbitrap Exploris for clinical research. Methods Proteins were directly extracted from dried blood spots (DBS) and analyzed by Thermo Scientific™ Orbitrap Exploris™ 240 with BioPharma Option coupled to Vanquish HPLC system. A 3.2 mm disc was punched from the DBS cards to 96 well plate and 50 uL water was added to re-hydrate. For protein precipitation, 150 uL acetonitrile was added and incubated at -20 ⁰C for 15 minutes. After micro-centrifugation, 160 uL of supernatant was removed and 70 uL water was added to resuspend. For LC-MS analysis, 10 uL of supernatant was diluted 8 times with mobile phase A and transferred to another 96-well plate. LC separation was performed on MAbPac RP column 4 um, 1 x 100 mm, and data-dependent acquisition mode was operated. Results With direct protein extraction from DBS, the entire sample preparation time is less than 1 hour. The MAbPac column showed its capability of separating between different chains with minor overlap between normal beta and HbS beta chains. Data processing was performed by ProSightPD, resulting in ∼45% sequence coverage for both normal Hb and HbS beta chains. The mass difference between normal beta and Hb S beta chains is about 30 Da, which results in about 1.6 m/z difference at a charge state of 19. This mass difference can often be hindered by other proteins or interferences from the matrix if the mass analyzer doesn’t have sufficient resolving power. Here we successfully applied Orbitrap resolution at 120,000 to isolate these similar masses for accurate variants or subunits identification including normal alpha, normal beta, Hb S, and delta chains. The confirmation of the delta chain can be beneficial since its abundance is usually less than 0.5% in adults, which shows potential for detecting low abundant variants using this method. Also, the calibration curve was generated to demonstrate the method's feasibility which is not a typical quantitation method for hemoglobinopathies. For quantitation, three most abundant m/z values from 2 charge states were added to TraceFinder for peak area extraction and normal Hb beta chain was used as an alternative internal standard for data processing. The 3-day inter/intraday analysis generated great reproducibility with CV < 10% and LOQ was determined to be 2.5 mg/mL with R2 values >0.994. Conclusions Successful demonstration of a single-step protein extraction directly from DBS and top-down analysis of Hb variant for clinical research.

Dried plasma spot as an innovative approach to therapeutic drug monitoring of apixaban: Development and validation of a novel liquid chromatography-tandem mass spectrometry method.

Apixaban, a direct oral anticoagulant drug (DOAC), typically does not require routine therapeutic drug monitoring (TDM), yet recent guidelines propose its use in specific clinical scenarios. While various antifactor Xa (anti-FXa) chromogenic assays serve as useful proxies for measuring plasma exposure to apixaban in emergencies, they lack specificity compared with chromatographic methods. This research project is intended to the development and validation of a standardized protocol of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conformity with the ICH guidelines M10 for the measurement of apixaban in both plasma and dried plasma spots (DPSs). Samples preparation included protein precipitation after the addition of a deuterated internal standard (IS), and the chromatographic separation was carried out on a Thermo Scientific™ Accucore™ Polar Premium column (50 mm × 2.1mm, i.d. 2.6 m). The newly developed LC-MS/MS method for apixaban mesurement from both plasma and DPS resulted linear over a wideconcentration range (31.25-500 ng/mL), accurate, and reproducible without matrix effects, allowing for specific and rapid quantification. Stability was assessed on quality controls and a real sample, allowing the setting up of a robust TDM protocol that was applied to five anonymized plasma samples obtained from adult patients undergoing apixaban treatment at steady-state. In conclusion our novel LC-MS/MS method is adequate for accurate apixaban quantitation from both plasma and DPS matrixes, andmay thus facilitate the guidelines suggested implementation of apixaban TDM, even in peripheral hospitals through shipment of DPS at reference laboratories.

The OrbiTOF mass analyzer: Time-of-flight analysis via an orbitrap quadro-logarithmic field with periodic drift focusing

Thermo Scientific™ Orbitrap™ analyzers represent a prominent class of high-resolution mass analyzer commonly used in life sciences, and for interrogation of complex samples. Injected ions, trapped within a quadro-logarithmic field, orbit a central electrode and oscillate up and down the axis. A new class of multi-reflection time-of-flight mass analyzer has been developed based on the Orbitrap field structure plus an additional series of periodic lenses wrapped around the central axis to constrain beam dispersion. The axial and angular velocity of the injected ions was balanced so that with each axial oscillation, the ions passed through the next lens in the series, to form a tightly folded 25-m long, 3-dimensional ion path, ending with ions striking a detector surface.Performance was interrogated via experiment and simulation. 70k resolving power was observed within the relatively compact analyzer, albeit at cost to transmission. A larger design with an integrated extraction trap and greater flight energy is discussed.

Evaluation of three different DNA extraction methods for the detection of Pepper yellow leaf curl virus (PepYLCV) by Polymerase Chain Reaction

Abstract Pepper yellow leaf curl virus (PepYLCV) from the genus Begomovirus infecting Chili pepper plant can cause significant yield losses. PepYLCV infection can lead the development of mosaic yellow, mottled pattern and yellowing leaves, which can be challenging in disease management and control. Developing effective detection methods for PepYLCV is a critical disease management and crop loss mitigation. The Begomovirus viruses have extremely low viral concentrations and are limited to the phloem and vascular system. DNA extraction is an important step in PepYLCV detection. This study aimed to evaluate the efficiency of three DNA extraction methods: phenol-based extraction, CTAB-based extraction, and the GeneJET Plant Genomic DNA Purification Kit (Thermo Scientific™). These methods were evaluated for their performance to identified the presence of PepYLCV DNA and provide accurate results for further analyses. DNA samples were extracted from two varieties including Red Habanero and F8 012328-6-2-1-1-3-1, and were subsequently amplified using Krusty Homer’s primers. The results showed that all three extraction methods possess the capability to identify PepYLCV. Based on the results, DNA extraction employing the CTAB-based method yields DNA with higher concentration and purity levels. Additionally, this method proves to be cost-effective and proficient in generating higher DNA concentrations.

Supernatant fluid from endobronchial ultrasound-guided transbronchial needle aspiration for rapid next-generation sequencing

There is an increasing demand to optimize the workflow and maximize tissue available for next-generation sequencing (NGS) for non-small cell carcinoma. We looked at transbronchial needle endobronchial ultrasound-guided bronchoscopy with transbronchial needle aspiration samples and evaluated the performance of supernatant (SN) fluid processed from a dedicated aspirate collected for NGS testing. Nineteen samples were collected and processed using a new workflow. Five aspirates were collected in formalin. One additional dedicated pass was collected fresh and centrifuged. The resulting cell pellet was added to formalin for cell block (CB) processing. DNA and RNA were extracted from concentrated SN for targeted testing using the Oncomine Precision Assay (Thermo Scientific, Waltham, MA). NGS results from the corresponding CB samples were used as "controls" for comparison. Thirty-one mutations were detected in SN (Table1). The most frequently mutated genes were TP53 (35%), EGFR (23%), KRAS (13%), CTNNB1 (6%), and ERBB2 (6%). There was 100% concordance between the mutations detected in SN and corresponding CBs with comparable variant allele frequencies. Turnaround time of NGS results was 1 day for SN compared to 4-10 days for CB. We were able to demonstrate the usefulness of SN for reliable rapid molecular results. We successfully incorporated the workflow for tissue handling and processing among our clinical, cytopathology, and molecular teams. Molecular results were available at the same time as the cytologic diagnosis, allowing for timely reporting of a comprehensive diagnosis. This approach is particularly useful in patients with advanced disease requiring urgent management.

Evaluation of Antibiotics by Disk Diffusion and Minimum Inhibitory Concentration Breakpoints in Urinary Tract Infections

Antibiotic resistance (ABR) has made it more challenging to treat uropathogenic organisms. It is impossible to compromise antimicrobial susceptibility testing (AST), which is essential and has a significant impact on infection treatment strategies. Although labor-intensive and technically challenging for everyday laboratory use, the agar dilution technique is appropriate for monitoring and assessing novel antimicrobials. Objective: To determine the minimum inhibitory concentration (MIC), Agar dilution technique and disk diffusion as susceptibility test methodologies. Methods: This study was carried out at Khyber Girls Medical College (KGMC) Peshawar. Keeping in view the Clinical and Laboratory Standards Institute (CLSI) guidelines AST was executed. BIOMÉRIEUX® API® kits and gram staining were utilized for identification of bacteria. The disk diffusion was performed using Thermo ScientificTM OxoidTM antibiotic discs of Co-trimoxazole, Levofloxacin, Nitrofurantoin and Fosfomycin. The MIC and zone of inhibitions for disk diffusion were noted according to the CLSI protocol. Results: 158 culture positive samples were isolated out of 680 total received. Esherechia Coli (E. coli) (74.1%) being the most isolated organism. In comparison of disk diffusion and agar dilution, categorical agreement for Levofloxacin, Cotrimoxazole, Nitrofurantoin and Fosfomycin were (82.28%, 72.15 %, 87.97% and 82.28%) respectively. Kappa coefficients of (0.64, 0.43, 0.57 and 0.37) (p < 0.0001) were calculated for Levofloxacin, Co-trimoxazole, Nitrofurantoin, and Fosfomycin respectively, revealing considerable level of agreement for these antibiotics. Conclusions: It was concluded that Agar dilution is more precise than disk diffusion but being more labor intensive and technical. Disk diffusion can still produce significantly accurate results with less resource consumption.

Enterobacter asburiae ST229: an emerging carbapenemases producer

Enterobacter asburiae, member of the Enterobacter cloacae complex (ECC) group, shows an increasing clinical relevance being responsible for infections like pneumonia, urinary tract infections and septicemia. The aim of the present study was the investigation of the genomic features of two XDR E. asburiae ST229 clinical strains co-carrying blaNDM-1 and blaVIM-1 determinants, collected in October 2021 and in June 2022, respectively. Two E. asburiae strains were collected from rectal swabs of as many patients admitted to the cardiopulmonary intensive care unit of Fondazione I.R.C.C.S. “Policlinico San Matteo” in Pavia, Italy. Based on the antibiotic susceptibility profile results, both isolates showed an XDR phenotype, retaining susceptibility only to fluoroquinolones. Both isolates shared identical resistome, virulome, plasmid content, and belonged to ST229, a rarely reported sequence type. They co-harbored blaNDM-1 and blaVIM-1 genes, that resulted located on transferable plasmids by conjugation and transformation. Moreover, both strains differed in 24 SNPs and showed genetic relatedness with E. asburiae ST709 and ST27. We described the first case of ST229 E. asburiae co-harboring blaNDM-1 and blaVIM-1 in Italy. This study points out the emergence of carbapenemases in low-risk pathogens, representing a novel challenge for public health, that should include such types of strains in dedicated surveillance programs. Antimicrobial susceptibility testing was carried out using Thermo Scientific™ Sensititre™ Gram Negative MIC Plates DKMGN. Both strains underwent whole-genome sequencing (WGS) using Illumina Miseq platform. Resistome, plasmidome, virulome, MLST, plasmid MLST and a SNPs-based phylogenetic tree were in silico determined.

A Study on the Efficacy of Autologous Platelet-Rich Plasma Therapy in Erosive Lesions of Oral Cavity

ABSTRACT Autologous platelet-rich plasma (PRP) enhances the recruitment, proliferation, and differentiation of cells involved in tissue regeneration. Platelets contain a number of growth factors (GFs) including platelet-derived GFs and transforming GF which stimulate the proliferation of mesenchymal cells and vascular endothelial GFs and fibroblast GF which stimulate new blood vessel formation. This study evaluates the efficacy of intralesional injection of PRP in erosive lesions of the oral cavity. Five patients who attended the dental outpatient department of a tertiary care hospital were selected irrespective of age and sex diagnosed with erosive lichen planus, pemphigus, and three with desquamative gingivitis. Initial assessment including blood investigations and evaluation of extent, severity, activity, and pain was done. Autologous was prepared from 30 milliliters of whole blood collected into a 350 ml blood collection bag after modifying the anticoagulant to whole blood ratio. A soft spin followed by a hard spin in Thermo Scientific™ Heraeus™ Cryofuge™ was used for PRP preparation. On the same day, 0.5 mL PRP was directly injected onto the base and edge of the erosions using a 31 gauge needle. Patients were followed up and evaluated weekly. In pemphigus and desquamative gingivitis, PRP therapy relieved pain, burning sensation, and bleeding which was proven from Visual Analog Scale score and severity score. No adverse reactions were noted in patients. Autologous PRP poses no risk of allergic reaction and natural collagen is formed in response to the presence of activated platelets. The use of PRP is safe, efficient, and economic. The inability to prevent recurrent lesions and the inability to heal the lesion completely are the major drawbacks.

Chemical investigation of biological trace evidence; toxicological screening of waste residues obtained from DNA extraction processes.

In a forensic scenario, if biological stains are found in very small quantities, these are usually collected for DNA analyses, considered essential for the forensic investigation and thus excluding possible investigations by other forensic disciplines as forensic toxicology.We developed an experimental study to evaluate the feasibility of analyzing DNA extraction residues obtained from DNA extraction procedures to perform toxicological analysis, with the aim to extract both genetic and toxicological information without affecting or compromising the genetic sample and/or DNA extraction.DNA extraction from four blood samples (fortified with 5 molecules of interest with a final concentrations of 1µg/mL, 100ng/mL, 10ng/mL and 5ng/mL, respectively) were analyzed with QIAGEN QIAmp® DNA Mini kit. Three waste residues collected from the DNA extraction were analyzed for the toxicological investigation via Solid-Phase Extraction and High-Performance Liquid Chromatography-Tandem Mass Spectrometry analyses (Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer). The analytical investigation revealed that our analytes of interest were detected in two different residues of the DNA extraction procedure, allowing both genetic and toxicological analyses without affecting the DNA identification. At last, the experimental protocol was applied to a hypothetical case, with encouraging results and allowing the identification of our molecules of interest.

ПОРОДНІ ОСОБЛИВОСТІ АЛЕЛЬНОГО ПРОФІЛЮ ГЕНІВ PIT1 ТА GH УКРАЇНСЬКОЇ АБОРИГЕННОЇ ЛЕБЕДИНСЬКОЇ ПОРОДИ КОРІВ

The preservation of genetic diversity is a paramount concern in addressing global challenges today. The foundation of biological diversity hinges on its genetic components, and the loss of species and genetic variability poses a genuine threat to the biosphere. This is because the sustainability of natural ecosystems and agroecosystems depends directly on their genetic adaptability to changing environmental conditions.
 In cattle, genes associated with milk yield play a crucial role, including genes related to milk proteins and hormones. Among these, the pituitary-specific transcription factor (PIT1) and growth hormone (GH) genes are of particular significance. In this study, we investigated the polymorphism of these genes in the Lebedyn breed of cattle.
 A total of 2 gene polymorphisms, PIT1 and GH, were examined through molecular and genetic research conducted at the genetics laboratory of the Institute of Animal Breeding and Genetics named after M. V. Zubets, part of the National Academy of Agrarian Science of Ukraine. The study utilized 32 DNA samples extracted from the venous blood of Lebedyn breed cows using the 'DNA Sorb-B' kit from AmpliSens. Genotyping was carried out through polymerase chain reaction (PCR-RFLP) analysis of restriction fragment length polymorphism, employing the “Tertsyk” amplifier for PCR. The resulting DNA restriction analysis products were assessed via electrophoresis in a 2–3% agarose gel containing ethidium bromide, with DNA fragment lengths determined using the GeneRuler TM 50 bp DNA Ladder and Thermo Scientific™ GeneRuler 1 kb Plus DNA Ladder markers.
 For the PIT1 gene analysis, a 451 bp amplified fragment was treated with the enzyme HinfI, yielding the following allele frequencies: A (0.38) and B (0.62). The heterozygous AB genotype was the most prevalent (0.45), while homozygotes for the A allele constituted 0.15 of the population. In the case of the GH gene fragment (223 bp), hydrolysis was performed using the restriction enzyme AluI. Analysis of allele frequency distribution indicated a high frequency of the L allele (0.85), with no animals exhibiting the homozygous GHVV genotype, and the GHLL genotype was present in 0.7 of the population.
 These findings reveal unique features of the allelic spectrum of the PIT1 (V-0.62) and GH (L-0.85) genes specific to the Ukrainian indigenous Lebedyn breed of cows. Selectionally significant genotypes include PIT1AA (15%) and GHLL (70%), while heterozygote carriers, which contribute to genetic diversity within the population, account for 45% and 30%, respectively.
 This study’s results hold particular value due to the alarming decline in the population of local indigenous breeds and the imminent threat to the preservation of agricultural genetic resources.

Forensic toxicological analyses reveal the use of cannabis in Milano (Italy) in the 1600's

In this paper, we reported the results obtained from toxicological investigations on bone samples collected from human remains of the 17th century in Milano (Italy). The aim of this study was to search for analytical signs of the administration of medical or recreative plants in the population of Milano during the 17th century.Nine femoral bone samples were extracted via Solid-Phase Extraction and analyzed via Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer.As a result, archeotoxicological analyses revealed the presence of two cannabinoids (Delta-9-tetrahydrocannabinol and cannabidiol) of the cannabis plant present in two out of nine bone samples (that correspond to 22% of the biological samples analyzed). The presence of these two alkaloids evidences the use of the cannabis plant in the Italian population during the 17th century. The documentation related to the pharmacopeia used to treat patients in the Ca’ Granda is still present in the archives of the hospital. However, this plant was not listed inside the pharmacopeia, suggesting that the plant was not administered as medical treatment at the time in the hospital.Thus, we hypothesize that the subjects under investigation used cannabis as a recreational substance, although other sources of exposure, such as self-medication, administration as a medical plant by other doctors outside of the Ca’ Granda, occupational and involuntary exposure, cannot be excluded.In conclusion, this study reports the first physical evidence of cannabis use in Modern Age in Italy but also in Europe.

B-307 Direct Quantitation of 77 Therapeutic and Clinical Toxicology Drugs in Dried Blood Spots Using the Fully Automated Transcend DSX-1 System

Abstract Background The dried blood spot (DBS) sampling technique is advantageous over the traditional liquid blood collection due to its minimal invasiveness, smaller sample volume, improved analyte stability, and ease of storage and transportation, resulting in its increasing usage in therapeutic drug monitoring and clinical toxicology. Here, we describe a fully automated workflow to rapidly extract and quantify a wide range of therapeutic drugs and drugs of abuse in DBS using the Thermo Scientific™ Transcend™ DSX-1 system, fulfilling the routine drug monitoring needs in the clinical laboratories. Methods Calibrators in blood are spotted on the DBS cards, analytes are extracted with the innovative flow-through desorption technology, and their corresponding internal standards (IS) are introduced via an automated IS addition module in the dried spot autosampler. The 2-dimensional TurboFlow technology allows the removal of interferences from the extracted samples before analytical separation. An integrated software controls every step of the sample desorption and separation. Analyte quantitation is performed on the Thermo Scientific™ TSQ Altis™ Plus triple quadrupole mass spectrometer, and the data is analyzed in TraceFinder™ software. Results A total of 77 therapeutic and clinical toxicology drugs from eleven classes, including anticonvulsants, antidepressants, antihistamines, antipsychotics, benzodiazepines, cocaine, dissociatives, opioids, and stimulants, are quantified in a single injection from DBS cards using a rapid automated method on a Transcend DSX-1 system. DSX-1 combines a dried spot module for direct analyte extraction with Transcend™ UHPLC for online sample separation using the TurboFlow technology. The method only takes 4.3 min from analyte extraction to MS detection. Good calibration curves with R2 > 0.98 are achieved using a weighting factor of 1/x, and the limit of quantification (LOQ) values are established with % RSD and % CV < 15, │% Diff│ < 20, and relative ion ratio < %20. The LOQ values are all in the low ng/mL levels, which largely meet the screening and confirmation sensitivity needs of analytical methodologies in routine clinical laboratories. Conclusion A fully automated LC-MS-based workflow is developed to rapidly and reliably quantitate 77 compounds therapeutic and clinical toxicology drugs in dried blood spots.

B-297 Effortless and Streamlined Workflow for Absolute Quantitation of Therapeutic Monoclonal Antibodies Using Promise Proteomics mAbXmise Kits and TSQ Altis Plus Mass Spectrometer

Abstract Background Laboratories continuously seek improved productivity and efficacy for clinical testing, ultimately impacting the turn-around time and sample throughput. Various efforts have been made including rapid and high-throughput testing, automation, and high-end instrumentation. This also applies to therapeutic drug monitoring (TDM) of therapeutic monoclonal antibodies (mAbs). In particular, mass spectrometry (MS) has gained significant popularity in clinical laboratories for TDM of mAbs due to its great versatility to measure such complex biological proteins qualitatively and quantitatively. Here we present the streamlined workflow for the absolute quantitation of therapeutic mAbs using the Promise Proteomics mAbXmise kits and the Thermo Scientific™ TSQ Altis™ Plus mass spectrometer. The mAbXmise kit is the first commercial ready-to-use kit that offers all the necessary reagents for the LC-MS/MS assay including the stable-isotope labeled (SIL) proteins of the target mAbs (SIL-mAbs). The TSQ Altis™ Plus mass spectrometer is the latest generation of the TSQ MS series, offering superior acquisition speeds, sensitivity, selectivity, and robustness for routine and high-volume targeted protein quantitation. Methods The experimental workflow followed the instructions provided by Promise Proteomics with minor modifications (refer to instructions manual—https://customer.mabxmise.com). The kits used in this study include pre-coated plates with a full-length SIL-mAb corresponding to each target mAb sequence as internal standards with purity >95% and isotopic corporation >98%. Thus, the ITDM1 kit contains 2 SIL-mAbs and the OTDM1 kit contains 7 SIL-mAbs pre-coated in each plate. The kits also provide buffer stocks, reagents, 6 calibration standards including zero, and 2 QC controls. Each kit provides 12 strips capable of 96 sample preparation. The calibrators and QC samples were added to the mAbXmise plate and subjected to immunocapture followed by trypsin digestion. LC-MS/MS was performed using a Thermo Scientific™ Vanquish™ Flex UHPLC system hyphenated to TSQ Altis™ Plus mass spectrometer. Results Highly comparable results were observed across six replicates for both OTDM1 and ITDM1 kits with R2 values >0.99 and % RSD of peak areas of peptides <15. After confirming the complete sample preparation of 96 samples, an evaluation of LC-MS analytical performance was performed. The results show great linearity with R2 > 0.998 over measured calibration concentration points for all the OTDM1 and ITDM1 target peptides, respectively. Also, the data support the reproducible LC-MS measurement of the entire plate providing % RSD <10. The accuracy of QC samples was determined to be between 94 and 103% for OTDM1 peptides with a precision of <7% CV and between 95 and 107 % for ITDM peptides with a precision of <9% CV. The response of CAL1 (LLOQ) was 5 times higher than CAL0 for all the target peptides with accuracy between 96 and 112% and precision of <11% CV. Additionally, extremely reproducible retention times were observed from all SIL-mAb peptides with RT difference ±0.03 min during the evaluation. Conclusion The workflow tested in this report generated sensitive and robust data with high confidence, emphasizing the capability of MS-based TDM of mAbs as routine testing.

B-100 Hbsag Performance Improvement of Novel MASTM Infectious Controls for Serology Diagnostic Tests

Abstract Background Hepatitis B is a liver infection caused by the Hepatitis B virus. While preventable by vaccination, Hepatitis B remains prevalent across the globe. While prevalence is low in many areas of the world, there are still regions in which the prevalence is considered to be high. Hepatitis B surface antigen (HBsAg) can be one of the first indicators of Hepatitis B infection, and it is a commonly screened diagnostic test to determine if a patient is immune, susceptible, or has acute or chronic HBV infection. While HBsAg is a critical marker in diagnosing a patient, it presents unique challenges. The Thermo Scientific™ MAS™ Omni™ Infectious BSI Positive Control Panel* is a multi-constituent IVD control containing anti-HIV-1/2, anti-HBc, anti-HCV, and anti-HTLV I/II antibodies, as well as Hepatitis B surface antigen (HBsAg) in a human plasma-based matrix. Development and Stability Studies were conducted to evaluate and ensure optimal analyte performance throughout the shelf-life duration. As this is a multi-constituent control, the HBsAg challenges must be adequately addressed to ensure the integrity and reliability of the results of all analytes. Two specific areas of focus are HBsAg instability at increased temperature and HBsAg-Fibrinogen interaction in plasma products. We report here the continued development and assessment of HBsAg in the MAS™ Omni Infectious Controls family. Methods The BSI Positive Control Panel was evaluated with IVD assays to assess Shelf-Life and demonstrate the effect of elevated temperature and fibrinogen on HBsAg. Shelf life of the products was determined by real-time, accelerated, open vial, and in-use stability monitoring. Additionally, open-vial stability monitoring demonstrates the relationship between HBsAg and fibrinogen within the control products. Between Month 0 and 12, samples were run neat without processing. At Month 12, samples were centrifuged to verify the validity of the open-vial stability claim. Results The evaluation of the BSI Positive Control Panel demonstrates the effects of temperature and fibrinogen on HBsAg. The Real-Time and Accelerated stability studies combine to demonstrate the effect of elevated temperatures on HBsAg within the multi-analyte control. The Open-Vial stability studies completed at 0 and 12 months combine to demonstrate the effect of fibrinogen on HBsAg within the multi-analyte control. Conclusions The MAS™ Omni Infectious control products will perform optimally provided the proper sample processing and storage conditions are implemented. Improper storage at elevated temperatures will result in degradation at an increased rate for the HBsAg present in the sample. Additionally, it is imperative that the proper raw materials are chosen in the production of the multi-analyte controls. When using plasma products, it is best to use defibrinated plasma. For extra precaution, raw material should be centrifuged and filtered prior to production to avoid degradation due to fibrinogen. Reliable, high-quality controls are vital in ensuring that clinical diagnostic assays are functioning properly. *Availability of product in each country depends on local regulatory marketing authorization status

B-099 Development and Improvement of Novel MASTM Infectious Controls for Serology Diagnostic Tests

Abstract Background Human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) remain major infections around the world. As not all viral infections are preventable, serological assays play a critical role in diagnoses, therapeutic monitoring, and treatment of these infections. The Thermo Scientific™ MAS™ Omni™ Infectious Controls are a family of controls containing single or multiple analytes in human plasma-based matrix designed to mimic patient samples in the assessment of serological assay performance. One of the greatest challenges within the field of Infectious Serology testing is method variation amongst the semi-quantitative assays. As an example, a result that is considered a low positive on one analyzer, may be considered a high positive on a different analyzer. Therefore, platform agnostic Quality Control material will provide evaluation among different methods. Quality Controls may also be developed to focus on certain platforms based on “fit-for-purpose” applications. We report here the development and performance evaluation of the MAS™ Omni Infectious Controls - For Use with Siemens Assays. Methods Two new MAS™ Omni Infectious Controls were developed and characterized to be added to the existing catalog of MAS™ Omni Infectious Control Products. MAS™ Omni Infectious BSI (Bloodstream Infectious) Positive Control Panel—For Use with Siemens Assays* is a multi-constituent IVD quality control containing anti-HIV-1/2, anti-HBc, anti-HCV, and anti-HTLV I/II antibodies, as well as Hepatitis B surface antigen (HBsAg) that was developed for the Siemens Assays platform. The new product has been adapted from the existing BSI Positive Control Panel* by increasing the titer of HBsAg while decreasing the titer of anti-HBc and anti-HCV. MAS™ anti-HBs Positive Control - For Use with Siemens Assays* was developed for the assessment of assay performance detecting anti-HBs on the Siemens Assays Platform. The new product has been adapted from the existing anti-HBs Positive Control by increasing the titer of anti-HBs. Development and performance evaluations were performed on SIEMENS™ and non- SIEMENS™ platforms. Shelf life of the products are determined by accelerated, real-time stability, open vial, and in-use monitoring. Results Two new MAS™ Omni Infectious Controls were developed and evaluated to be specifically used on the Siemens Assays Platform. Both products demonstrated acceptable performance on the SIEMENS™ Centaur™ and Abbott™ Architect™. Stability studies demonstrated a minimum of 24- or 12-months shelf life, 30 or 60 days of open vial stability, and 8 hours of in-use stability when stored at 2–8°C, respectively. Additionally, the studies demonstrate a basic understanding of method variation within Infectious Serology test methods. Conclusions Two new MAS™ Omni Infectious Control products that mimic patient samples have been developed for the assessment of assay performance on the Siemens Assay Platform in the clinical laboratory for anti-HBc, anti-HCV, and Hepatitis B surface antigen (HBsAg). These reliable and stable control materials will better support the assessment of serological assay performance on the Siemens platforms. *Availability of product in each country depends on local regulatory marketing authorization status