bluuebunny/crossref_metadata_2025_split huggingface dataset:
B-260 Innovative One-Step Extraction of Exosomal micro RNA Without Isolating Exosomes
Authors: Bae M , Shin S 
Published: September 2023

Abstract

Background
Extracellular vesicles (EVs) are receiving great attention as biomarkers in liquid biopsies since they are involved in physiological and pathological processes. It is important to obtain a high-yield of nucleic acids because it is possible to prediagnose through the exosome.

Methods
Here, we report an efficient one-step method for extraction of miRNAs by clustering EVs. When cationic salt is added to samples, anionic EVs tend to form clusters and precipitate within a short time. After centrifugation and adding lysis buffer, miRNA can be extracted with a spin column. Commercial methods were compared to determine the yield of the precipitation method using cationic salt. In addition, we compared with two reference methods to determine the miRNA extraction efficiency. The first one is consisted of two-step method of extracting miRNA from isolated exosome (2 step), which is a general method, where as the second reference method is a commercial product consisting of 1-step extracting miRNA from biofluids.

Results
Cluster precipitation efficiency using cationic salt was identified through cluster image, SEM, and Western blot. The present method for extract exosomal miRNA has an EV isolation efficiency of more than 3 times compared to the commercial EV isolation methods. In addition, it has excellent exosomal miRNA extraction efficiency from various biofluids such as Saliva, Urine, and Plasma, and can be used as a sample of breast cancer patients.

Conclusion
These results might be contributed by the sequence of precipitation of exosomes, exosome lysis, and nuleic acid extraction. The rapid and efficient method for extracting exosomal nucleic acids will make an innovative contribution to clinical applications.
B-262 Evaluation of the Genotypic Profile of the c.202 G&gt;A Mutation in the G6PD Gene in Brazil
Authors: Silva J D , Rios J S , Zauli D A 
Published: September 2023

Abstract

Introduction
Glucose 6 phosphate dehydrogenase (G6PD) is an enzyme found in the cytoplasm of all human cells and plays an important role in preventing cellular damage to reactive oxygen species (ROS). G6PD deficiency is inherited in an X-linked recessive genetic disorder that can destroy red blood cells (hemolysis) leading to acute hemolytic anemia during periods of increased ROS production. The disease prevalence is more frequent in male individuals. To date, more than 400 G6PD variants have been identified, and molecular studies have shown that G6PD deficiency can be associated with different mutations. The c.202 G &gt; A mutation (rs1050828; NM_000402.4) was described as the most frequent. The rapid and accurate diagnosis of this condition is extremely important in neonatal screening and cases of diagnostic suspicion. Therefore, this study aims to describe the genotypic profile of the c.202 G &gt; A mutation in the G6PD gene in Brazilian samples by two molecular assays.

Methods
Genomic DNA samples from 130 individuals previously genotyped by Restriction Fragment Length Polymorphism PCR were used for validation. DNA isolation was performed in MagNA Pure 24 Total NA Isolation Kit at the automated platform MagNA Pure 24 System (Roche Diagnostics, Basel, Switzerland), according to manufacturer instructions. Genotyping for rs1050828 was performed using TaqMan SNP Genotyping Assays (Thermo Fisher, Foster City, CA) at 7500 Fast Real-Time PCR System (Thermo Fisher, Foster City, CA), according to manufacturer instructions. The results were analyzed using TaqMan Genotyper Software version 1.0.1.

Results
This study evaluated 130 blood samples, of which 81 were men (62.3%) and 49 were women (37.7%). Male carriers of rs1050828 A genotype (hemizygotes) predominated (56/81, 69.1%) and the remaining carriers for allele G (wild type) were 25/81 (30.9%). While female carriers of rs1050828 GG (wild type) predominated (39/49, 79.6%), heterozygotes were 5/49 (10.2%), and homozygotes also were 5/49 (10.2%). The number of male individuals with the presence of the mutation in hemi/homozygotes was considerably higher when compared to female individuals. This is explained by the fact that the mutation is inherited in an X-linked recessive manner. The results showed agreement and reproducibility of 100% for both methodologies. However, the qPCR assay sensibility and efficiency (&gt;99%) were higher than the RFLP PCR assay.

Conclusions
Here, we described an efficient molecular method to detect the mutation c.202 G&gt;A in the G6PD gene. The frequency of c.202 G&gt;A described in this study was similar to that observed in others population studies in Brazil. Detection of the mutation target requests an additional evaluation of the G6PD activity level. Obtained results should be evaluated in association with the clinical aspects. The absence of the c.202 G&gt;A in the G6PD gene does not exclude the existence of other mutations in the same region.
B-276 Preservation of Proteomic Inflammatory Biomarkers signal in a Saliva Multiomic Buffer at Room Temperature
Authors: Jensen M M , Maecker H , Nguyen T , Vu D , Vigerust D , Gupta R 
Published: September 2023

Abstract

Background
Saliva represents an attractive biospecimen as it is pain-free, simple for at home collections, and patients can provide multiple samples over time to provide time resolved data. However, few diagnostic tests have been developed using saliva due to poor stability and comparatively low concentration of analytes. Stabilization of biomarkers at ambient temperature in saliva would enable labs to take advantage of the benefits of saliva sample collections, while minimizing the risks posed by the degradation of already scarce analytes.

Methods
Matched samples of neat serum, saliva, and saliva collected with Saliva Multi-omic Device (SMD) preservative were collected from 30 volunteers (15 male & 15 female) ages 18–64 with no known morbidities. The Olink Inflammation panel was used to examine 92 inflammation-related proteins. The method utilized proximity extension assay technology, wherein antibody probe pairs reacted with their respective target proteins to generate a polymerase chain reaction target sequence through proximity-dependent DNA polymerization. This sequence was measured using real-time polymerase chain reaction and normalized in two steps, producing a semi-quantitative Normalized Protein eXpression (NPX) unit. To evaluate analyte stability specimens were measured initially and then again after 24 h at room temperature (23 ± 2°C).

Results
Protein expression (NPX) units for 36 of the 92 inflammatory markers had higher concentrations in saliva compared to serum. Serum (78/92) and saliva 78% (72/92) showed similar coverage for the number of markers in the panel where at least 75% of subjects had values exceeding the limit of detection (LoD). SMD preservation buffer improved the average stability of inflammatory protein biomarkers by 19.6% compared to neat saliva and 10.2% compared to serum over 24-hours at room temperature. However, the relative stability of individual between protein markers [DV1] [HM2] varied. For proteins in SMD buffer, initial concentrations of analytes were highly predictive [HM3] of values 24 h later, having Pearson correlation coefficients &gt; 0.7, for 23 of the analytes and greater than 0.5 for 47. The change in protein marker in serum over time was only moderately predictive of the protein’s stability in saliva with or without preservative, having correlations of 0.33 ± 0.20 and 0.47 ± 0.19 (mean ± standard deviation), respectively.

Conclusion
This study demonstrates potential utility of saliva in proteomic applications utilizing the Olink platform. The SMD preservation buffer stabilized inflammatory biomarkers in saliva samples likely through protease inhibition and antimicrobial action. Preservation of self-collection salivary samples by subjects without cold storage enables noninvasive sampling and reliable measurements even when samples are collected by users at remote locations or under challenging conditions.
B-278 Pilot Clinical Study of the CarePoint Assay
Authors: Briggs1 J S , Brooks-Starks A H , Elenberger T J , Brenneman R , Scarpato K R , Chang S S , Bassett J C , Linehan J 
Published: September 2023

Abstract

Background
An estimated &gt;710 000 individuals live with bladder cancer in the United States, with approximately 82 290 new cases expected this year (2023). Recurrence rates for bladder cancer are high: approximately 65% of patients with non-invasive or in situ tumors and 73% of patients with more advanced disease experience recurrence within five years. Consequently, patients treated for bladder cancer require prolonged surveillance to safeguard against tumor recurrence. The gold standard of surveillance, cystoscopy—an uncomfortable, expensive, and invasive procedure—is recommended every three to 12 months. Consequently, there is a need for an inexpensive, effective, noninvasive diagnostic tool as an alternative to cystoscopy.
The CarePoint Assay (CPA) is a 30-minute electrochemical immunoassay for the detection of disease-state biomarkers in human urine. The analytical performance of CPA has been evaluated for method comparison with a three-hour sandwich immunosorbent assay (ELISA) for bladder tumor antigen (BTA), a diagnostic biomarker for bladder cancer.

Method
Receiver operating characteristic (ROC) curves were calculated by simple logistic regression with Prism9 (GraphPad) using patient urine samples across multiple medical centers (n = 51), comparing BTA concentration (continuous variable) with high-grade tumor status (binary variable). Patient urine samples were evaluated by CPA using commercial potentiostats for comparison with a sandwich ELISA. The data was analyzed using simple linear regression.

Results
Patient urine evaluated by CPA was interpolated by simple linear regression using a three-point calibration with standard spiked into urine pooled from healthy donors. Data produced by sandwich ELISA was interpolated by five-parameter nonlinear regression using a 12-point standard curve. Both methods showed good correlation: (r) = 0.93. Simple logistic regression comparing analyte concentration to high-grade tumor status produced a sensitivity and specificity of 52% and 88% with an AUROC of 0.71 for the CPA, and a sensitivity and specificity of 72% and 77% with an AUROC of 0.77 for the sandwich ELISA. Sensitivities and specificities were calculated using Youden’s J statistic.

Conclusions
We established proof of concept for a rapid, electrochemical immunoassay for a diagnostic biomarker of bladder cancer. The CPA correlated well with sandwich ELISA, an established method for measuring biomarkers in bodily fluids. Future work will focus on refinement of the CPA architecture for point-of-care use and subsequent evaluation of analytical performance.
B-289 Acute Kidney Injury Urinary Biomarker, Neutrophil Gelatinase-Associated Lipocalin (NGAL) Analytical Performance Evaluation on Roche Cobas c502 Chemistry Platform
Authors: Samara V , Buggs V , Song L 
Published: September 2023

Abstract

Background
Acute kidney injury (AKI) is a sudden and serious kidney damage condition that affects more than 1.2 million people every year. KDIGO guidelines suggest serum creatinine increase as a diagnostic marker for AKI but elevation of serum creatinine levels can take up to seven days. Neutrophil gelatinase-associated lipocalin (NGAL) has been identified as an early tubular injury biomarker for AKI that is detected within three hours of AKI patients’ urine. In this study, we evaluated technical performance of BioPorto NGAL assay on Roche cobas c502 chemistry analyzer.

Methods
BioPorto’s (Hellerup, Denmark) particle enhanced turbidimetry immunoassay (PETIA) NGAL assay was installed on cobas c502 chemistry analyzer (Roche, Indianapolis, U.S.A) following vendor-specific application parameters with few modifications. Linearity was determined with BioPorto’s linearity material and verified by serial dilutions of a high NGAL patient urine sample. Limit of quantitation was determined in urine samples with values 20, 25 and 35 ng/mL, run in duplicate for 10 days. Precisions were determined by running urine samples containing 100 ng/mL and 400 ng/ml NGAL 20 times for within-run precision and twice a day × 10 days for between-run precision, respectively. To determine the accuracy of the NGAL method on cobas 502, 40 sample results (provided by the vendor) were compared with those measured on Siemens Atellica using BioPorto’s NGAL assay. Reference interval verification was carried out in 40 urine samples collected from healthy adult volunteer-donors.

Results
The linearity was found to be excellent from 0–3000 ng/mL. Limit of quantification was found at 26 ng/mL. Precision results are in the table below.
Method comparison showed a slope of 0.90 and intercept of 32.9. The reference interval was verified to be &lt;50 ng/mL (in accordance with current NGAL reference cut­off for AKI diagnosis).

Conclusion
Performance of BioPorto’s NGAL PETIA assay was validated in urine on cobas c502 chemistry analyzer.
B-300 Importance of Clinical Chemistry and Metabolism Parameters for the Management of Patient With Chronic Nitrous Oxide Intoxication
Authors: Grzych G , Gernez E , Marchand D , Deheul S , Zerimech F , Masso V , Bennis A , Diesnis R , Joncquel M , Kim I , Niguet J , Guichard J , Girot M , Tard C 
Published: September 2023

Abstract

Background
Recreational use of nitrous oxide (N2O) has increased exponentially recently and leads to severe neurological disorders. A recent report from European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) showed that the recreational use of N2O is a growing and became a major health issue in Europe. For biological monitoring, serum or urine N2O assays are not routinely performed because they do not allow to ensure a real exposure due to the very short half-life of this gas in the body. Functional disorders of vitamin B12 metabolism, characteristic of N2O intoxication, could be explored by two markers: homocysteine (Hcys) and methylmalonic acid (MMA). We experimented here, management of N2O abused patient thanks to functional markers as Hcys and MMA.

Methods
We collected clinical and biological data from patients with declared chronic N2O consumption. The patients treated in Lille University Hospital between september 2020 and december 2022 were included in the study. The clinical evaluation included the Peripheral Neuropathy Disability (PND) score which was evaluated by a neurologist.

Results
Our study included 202 patients with N2O abused, Globally, plasma functional markers concentration are increased in case on consumption (Table). We noticed that clinical sign of patient evaluated by PND score correlated with plasma MMA (P-value &lt; 0.01) but not with plasma hcys (P-value &gt; 0.05).

Conclusion
The use of functional markers as Hcys and MMA makes it possible to follow the metabolic disturbances of nitrous oxide abused patients and evaluated clinical outcomes. However, in view of the thrombotic risk related to high Hcys and the need to quickly adapt the nutritional treatment, biological and in particular Hcys, current reporting time is not satisfactory using mass spectrometry method. Hence, our medical laboratory aim to improve our method evaluating immunoassay techniques in order to reduce the turnaround time while maintaining analytical reliability.
B-305 A Novel Dilute-n-shoot Method for Quantitating Isotonitazene and Desmetonitazene in Human Urine By LC/MS
Authors: Simms P J 
Published: September 2023

Abstract

Background
Isonitazene is a benzimidazole derived opioid analgesic drug related to etonitazene, which has been sold as a designer drug. According to doctors isotonitazene has the potency of 20–200 times of fentanyl. The side effects of isotonitazene are like fentanyl and has been linked to many fatalities in Europe and the US. It has led to a number of overdoses through secondhand contact, by smelling or coming in contact with skin. It is slowly replacing fentanyl as the deadliest street drug in the US. We developed a dilute-n-shoot method that would test for isotonitazene and its metabolite metodesnitazene in human urine. This method used filtration to remove unwanted particulates and protein. It reduces the number of steps needed to prepare samples for liquid chromatography/mass spectrometry analysis compared to methods that require centrifugation. This assay allows for easier training of staff. In addition, it is easily adaptable to increase sample load.

Method
Isotonitazene and metodesnitazene urine samples were prepared for LC/MS analysis by diluting them 1:20 in 0.1% formic acid in water. These samples were spiked with internal standard and filtered through a 0.2 μm Captiva 96-well filter plate. Isotonitazene and metodesnitazene concentrations were measured using LC/MS in the positive ion mode. The separation was obtained using a phenylhexyl UPLC column, 50 × 2.1 mm, 1.8 μm, along with a 50:50 acetonitrile/methanol/water formic acid gradient mobile phase.

Results
The separation was completed in a proximately 4.5 min with good resolution between isotonitazene and metodesnitazene. Validation studies demonstrated that this method was linear from 2 to 100 ng/mL, with r2 ≥ 0.990 for both isotonitazene and metodesnitazene. Interday and intraday precision for both analytes were acceptable, with CV values &lt; 5.0%. Accuracy measurements at 5, 25 and 80 ng/mL gave results where the percent difference was ≤20% for the 4 ng/mL concentration and ≤15% for the 25 and 80 ng/mL. A study of interfering compounds showed that there were no interferences that would affect the accuracy of isotonitazene and metodesnitazene. Recovery studies showed that &gt; 90% of each analyte was recovered from the filtration step. The LOQ was determined to be 2 ng/mL and the LOD was determined to be 0.5 ng/mL. The reportable range was determined to be 2 to 200 ng/mL. The results of this validation will be discussed in detail in this poster.

Conclusion
The method was linear and precise for both analytes over a range of 2 to 100 ng/mL. The method was reproducible and easy to use. Isotonitazene and metodesnitazene could easily be quantitated in human urine using this method.
B-338 A Clinical Research LC-MS/MS Method for the Analysis of Immunosuppressant Drugs in Whole Blood Using Capitainer B Devices
Authors: Balloch S , Calton L , Hammond G 
Published: September 2023

Abstract

Background
Traditional laboratory analysis of the immunosuppressant drugs cyclosporine, everolimus, sirolimus and tacrolimus is well-established in clinical research. However there remains a need for individuals to undergo an invasive, time-consuming and disruptive process under the supervision of trained staff in order to collect a sufficient volume of whole blood for laboratory analysis. A reliable, remote sampling method may find utility in a clinical research setting. Here we describe the use of Capitainer® B qDBS devices to obtain analytically sensitive, precise and accurate data for cyclosporine, everolimus, sirolimus and tacrolimus analysis using small sample volumes. The Waters ACQUITY™ UPLC I-Class with Xevo™ TQ Absolute mass spectrometer was used to analyze these samples.

Methods
An in-house laboratory developed LC-MS/MS method to analyze all four immunosuppressants in a single ruin was developed. Waters MassTrak Immunosuppressant Calibrator and Control Sets (IVD) and whole blood External Quality Assurance samples (LGC, Bury, UK) were used in conjunction with Capitainer® B qDBS devices to assess the performance of the method. Samples were collected using Capitainer® B qDBS devices, and the sample (10 µL) extracted using solvent containing internal standards. A water/methanol/ammonium acetate gradient was used with a Waters C18 HSS SB column on a Waters ACQUITY™ UPLC™ I-Class and Xevo TQ Absolute Mass Spectrometer operating in positive electrospray ionization mode with run time of less than 2 min.

Results
Analytical sensitivity of the lowest calibrator at 1 ng/mL for everolimus, sirolimus and tacrolimus and 25 ng/mL for cyclosporine was demonstrated with S/N (PtP) &gt; 10 across five analytical runs. We successfully demonstrated linearity of cyclosporine from 25–1500 ng/mL and everolimus, sirolimus and tacrolimus from 1–30 ng/mL, with r2 &gt; 0.995 over five analytical runs. Total precision and repeatability across the four immunosuppressants (2, 8 and 22 ng/mL for everolimus, tacrolimus and tacrolimus; 150, 400 and 900 ng/mL for cyclosporine) with five replicates over five analytical runs (n = 25) was ≤15% CV. External quality assurance samples for all drugs met the scheme acceptance criteria, with mean bias ≤15% CV.

Conclusion
Using Capitainer® B qDBS devices and sample small volumes (10 µL) of whole blood, an in-house laboratory method was used to meet validation goals for analytical sensitivity, linearity, precision and accuracy for cyclosporine, everolimus, sirolimus and tacrolimus. Furthermore, the advantages conferred by volumetric absorptive microsampling, notably removing the requirement for travel and a venous blood draw and facilitating home sampling, render this technique applicable to clinical research. For Research Use Only. Not for Use in Diagnostic Procedures.