bluuebunny/crossref_metadata_2025 huggingface dataset:
B-221 Combined RT-PCR and Whole Exome Sequencing Pharmacogenetic Testing in Polypharmacy Patient: A Case Report
Authors: Rubio Prieto J L , Segura E , Pérez López E , Cordero Matia E , Rodríguez Rodríguez A , García de Veas Silva J , Guerrero J , Macher H
Published: October 2024

Background

Pharmacogenetics has become one of the main branches of Precision Medicine owing to its usefulness for patients’s optimal therapeutic management. Notwithstanding, pharmacogenetic testing results must be taken carefully, specially in multimorbidity patients with a variety of drugs prescribed. The aim of the present work is to introduce a combined RT-PCR and Whole Exome Sequencing Approach for pharmacogenetic testing in a patient with multiple drug-related adverse events.

Case Background

A 67 years old female was derived to our Clinical Chemistry Unit for pharmacogenetic testing due to multiple drug-related adverse events: nifedipine-voriconazole interaction, voriconazole-related neurological toxicity and moxifloxacin-related dizziness. Aditionally, she had been receiving morphine, corticoid for her reumatoid arthritis and antituberculous therapy (r isoniazid / pyrazinamide / rifampin and ethambutol).

Methods

Patient’s DNA was extracted from a blood sample and undergone by pharmacogenetic testing in two parallel ways: -A first genotyping assay was carried out using Taqman® OpenArray™ PGx Express 120 panel (ThermoFisher™), according to manufacturer’s instructions.

-Simultaneusly, in order to cover possible copy number variants and SNPs not covered by our RT-PCR array, a whole exome sequencing run was performed on IlluminaTM NextSeq 1000. Sample library was prepared applying a custom GRCh38 pipeline.

Results

Relevant variants related to patient’s therapy found by RT-PCR are shown in table 1.

SNPs genotyped by RT-PCR were confirmed by NGS. Moreover, whole exome sequencing revealed a deletion in exon 5 of SLCO1B1 gene, which corresponds to SLCO1B1*49 (loss of function); and some variants in NAT2 that may cause toxicity in antituberculous treatment (diplotype NAT2*6C/*7C).

Conclusions

In those challenging cases that involve multimorbidity and polypharmacy patients, pharmacogenetic testing must be approached taking into account variants that are not covered by the analytical method routinely used and the possibility of complement it with alternative proccedures.
B-222 Receptor-donor CYP3A5 and CYP3A4 genotype testing in liver transplant patients and its influence in tacrolimus blood levels: a retrospective study
Authors: Rubio Prieto J L , Macher H , Gómez Bravo M Á , García de Veas Silva J L , Suárez Artacho G , Rodríguez Rodríguez A , Sánchez Pozo C , Guerrero J , Rubio A
Published: October 2024

Background

Optimal immunosupression is crucial for transplant success. In orthohepatic transplants, blood target levels of tacrolimus, when coadministred with corticoids and mycophenolic acid, are between 6 and 10 ng/mL during the first three months post-transplant. Notwithstanding, achieving tacrolimus concentrations into this range may be tough because of factors such as liver function status, age, sex, drug-drug interactions, etc. In this way, pharmacogenetic testing of CYP3A5 and CYP3A4 genes have demonstrated to be useful in tacrolimus dosing optimization. Nonetheless, there is no evidence enough for pharmacogenetic based adjusting in liver transplant, specially if receptor and graft’s genotypes differ in these loci.

The aim of the present study is to find out how the receptor and donor genotypes in CYP3A5 and CYP3A4 genes affect tacrolimus pharmacokinetics.

Methods

Genotypic profiling of 108 receptor-donor's pairs biopsies gathered from patients who were underwent hepatic transplant from 2012 to 2019 were carried out using Taqman® OpenArray™ PGx Express 120 panel (ThermoFisher™). We focused on CYP3A5*3, *6 and *7; and CYP3A4*1B, *3 and *22 alleles. Next to pharmacogenetic testing, each patient was classified as very poor, poor, intermediate, rapid or ultrarapid metabolizer according to their own genotype, graft’s genotype or both of them. Patient’s initial dose, at seven days after treatment start and after discharge of tacrolimus and their trough concentrations (C0) were collected from their electronic health records.

Tacrolimus levels were measured by affinity chrome-mediated immunoassay. C0 was normalized by weight-adjusted dosage (C/D ratio); then, median C/D ratios between different metabolic profiles were compared through Kruskal-Wallis test.

Results

When patients were classified based on donor’s genotype isolated or combined with receptor’s genotype, lower C/D ratios were found as metabolizer ability increases (p=0.045 for donors and p=0.027 for combined genotypes, respectively). No statistically differences were found for C/D ratios at seven days and discharge, though that differences were almost significant for donor’s genotypes (p=0.06 in both cases).

Conclusions

These results show that previous information about both patient’s and specially donor’s CYP genes genotype may improve initial immunosupression and avoid tacrolimus induced toxicity. Pharmacogenetics has been currently proposed as a guide for tacrolimus treatment of some solid organ transplants as kidney transplanted patients. Better understanding of how receptor and donor’s CYP genotype affects its pharmacokinetics, along with liver’s damage markers and other genetic polymorphisms will allow clinicians, in the era of personalized medicine, to improve immunosupressive management.
B-228 Performance Comparison of three viral RNA pathogens in MK Buffered Solution for Diagnostic Based Detection using quantitative PCR vs droplet digital PCR
Authors: Casto M , Hicks R , Brazeau D , Karamchi M
Published: October 2024

Background

Droplet digital PCR (ddPCR) by partitioning a single reaction into many thousands of nano-droplets allows for the absolute quantitation of a given DNA/RNA target and represents the gold standard for the identification and quantification of pathogens in clinical and environmental samples. While nucleic acid based molecular diagnostics have become fundamental for the accurate and rapid quantification of specific pathogens, these instruments are still based upon quantitative PCR (QPCR) assays. The goal of this study was to assess the compatibility of a MK Buffered solution with three different RNA viruses combined into two standard clinical negative matrices and compare performance of ddPCR and QPCR for all three pathogens.

Methods

Serial dilutions of heat-inactivated Sars-CoV-2 (NR-52286), Influenza A (ATCC VR-1469) and RSV-A (ATCC strain VR-26) were combined into a single tube and added to two confirmed clinically negative matrices, Nasal and Sputum (Lee Biosolutions, Maryland Heights, MO). Serial dilutions of the combined pathogens were added to sample tubes containing 500 µL of MK buffered solution yielding final concentrations of each pathogen over 4 orders of magnitude in each clinical matrix. For each sample 300 µl was processed for RNA isolation using standard RNA isolation kits (Zymo Viral RNA Kit). For ddPCR samples were quantified for all three pathogens using a BioRad® Droplet Digital PCR System (QXD200). For QPCR, samples were quantified using Quantitative real-time PCR using Promega GoTaq® Probe 1- Step RT-qPCR System. The same TAQMan® probes specific for each pathogen were used for both systems.

Results

Yields of all three viral RNAs were significantly lower from sputum samples as compared to nasal samples. For all three pathogens the ddPCR assay was an order of magnitude more sensitive than observed with QPCR. QPCR and ddPCR values showed a significant linear relationship for all three pathogens (Sars-CoV-2, r2 = 0.984; RSV A, r2 = 0.814; Influenza A, r2 = 0.985).

Conclusions

QPCR and Droplet digital PCR accurately quantified concentrations of all three RNA pathogens in both clinically negative nasal and sputum matrices across four orders of magnitude, though detected yields were significantly lower in samples spiked into the sputum matrix. For all three pathogens there was a significant linear relationship with decreasing concentration. ddPCR was an order of magnitude more sensitive than QPCR for all three pathogens.
B-254 Analytical and Clinical Performance Evaluation of Plasma pTau217 in a Clinical Diagnostic Laboratory
Authors: Encarnacion M , Kumar P , Mousavi A , Hallett K , Biehl D , Pflueger S , Mammel A , Frykman H
Published: October 2024

Background

Blood-based biomarkers provide an accessible and cost-effective tool for diagnosing Alzheimer’s disease (AD). Among these biomarkers, phosphorylated tau 217 (pTau217) shows promise for both AD diagnosis and differential diagnosis, as well as for facilitating access to emerging disease-modifying therapies. The ALZpath SIMOA pTau-217 v2 assay is a digital immunoassay designed for quantifying pTau-217 in human EDTA plasma samples. This study assesses the analytical performance of plasma pTau217 assays as a Laboratory Developed Test (LDT) appropriate for diagnostic application.

Methods

In this analytical study, we evaluate accuracy, precision, detection limits, linearity, and analyte stability. Plasma pTau217 levels were measured using the ALZpath pTau217 v2 assay on a single Quanterix HD-X SIMOA Analyzer platform at BC Neuroimmunology Lab. Inc. Vancouver, British Columbia, Canada. As a clinical testing laboratory, our validation procedures are in accordance with CAP and CLIA requirements, with a primary focus on adhering to CLSI guidelines for both analytical and clinical performance testing. To determine a reference range and cut-off, samples of Amyloid PET-confirmed cases (n=263) and healthy subjects between the ages of 55 and 95, with a roughly equal ratio of males to females (n=219), were tested.

Results

The ALZpath pTau217 assay demonstrates an analytical measurement range of 0.0046 - 3.4056 ng/L, with a limit of blank (LoB) of 0.0019 ng/L, a limit of detection (LoD) of 0.0046 ng/L, and a limit of quantification (LoQ) of 0.03 ng/L. Linearity spans from 0.06 to 1.75 ng/L, with repeatability showing intra-laboratory precision above LoQ ≤ 20% CV. Stability is maintained at 4⁰C for ≤ 1 week, at room temperature for ≤ 72 hrs, at -20⁰C for ≤ 2 weeks, and at -70⁰C for ≤ 3 weeks, with freeze/thaw cycles tolerated up to 6 times. Moreover, interference is not detected at the maximum concentrations tested for albumin, unconjugated bilirubin, biotin, hemoglobin, creatinine, and heterophilic antibodies. The reference range was determined using the 95% reference interval, resulting in 0.398 ng/L. ROC analysis yielded an AUC of 0.932 with a Youden index of 0.338 ng/L. At the Youden index, sensitivity was 90.0% with a specificity of 85.6%. Specificity reached 95.5% at the upper cut point of 0.471 ng/L, while sensitivity was 94.8% at the lower cut point of 0.242 ng/L.

Conclusions

The outstanding analytical performance of pTau217 supports its adoption as a reliable screening and diagnostic tool in clinical practice, aiding clinicians in the accurate diagnosis of AD.
B-266 Therapeutic Drug Monitoring of Natalizumab and Anti-Natalizumab Antibodies
Authors: Chun K , Bauer B , Bastidas M , Zikry M , Yang J
Published: October 2024

Background

Natalizumab (NTZ) is a monoclonal antibody directed against the α4-integrin subunit of α4β1 and α4β7 integrins. NTZ is used to treat relapsing and remitting forms of multiple sclerosis. Although a majority of patients on standard dosing (intravenous 300 mg, every 4 weeks) reach therapeutic drug concentrations, about 10% of patients will eventually fail NTZ therapy due to the development of anti-NTZ antibodies. NTZ and anti-NTZ antibody concentrations in serum are used together to provide patient-specific pharmacokinetic and immunogenic assessment to aid in patient management.

Methods

Serum measurements of NTZ and anti-NTZ antibody levels are performed by lab developed electro-chemiluminescent immunoassays where assay design and principle are equivalent to biologic therapeutic drug monitoring described in our previous publication. The natalizumab drug assay is an immunoassay validated to measure free drug (pharmacodynamically active, ADA-unbound) in serum. The anti-natalizumab antibody assay utilizes a solution phase bridging method designed to measure total binding antibodies (including IgM & IgG subtypes). All anti-natalizumab antibody positive samples are confirmed for drug-specificity by an additional signal suppression step. The anti-natalizumab antibody assay is quantitative and drug-tolerant, meaning that circulating drug in patient serum does not interfere with anti-NTZ detection and quantitation.

Results

Here, we report concomitant measurement of NTZ and anti-NTZ antibody in 160 patient serum samples. Positive anti-NTZ levels from 24 to 235 ng/mL were found in 7.5% (12/160) patients. Anti-NTZ-free samples (148/160) had NTZ drug levels ranging from undetectable (<1.0) to 85 ug/mL. The mean and median drug level in 90 samples (results >1.0) was 10.8 and 2.7, respectively. 61/90 samples had therapeutic drug concentrations (at least 2.0 ug/mL). In the presence of anti-NTZ, 67% (8/12) had undetectable drug level and (4/12) had mean and median drug levels of 1.2 and 1.3, respectively, which is 9- or 2-fold lower than the mean and median drug levels of the anti-NTZ Ab -free group.

Conclusions

Low serum NTZ and high anti-NTZ are associated with loss of clinical efficacy, while elevated NTZ may increase progressive multifocal leukoencephalopathy risk. Assays to measure biologic drugs and their anti-drug antibodies in patient serum may be useful tools in dose optimization and patient management.
B-267 Validation of the Newly Developed Fully Automated Chemiluminescent Enzyme Immunoassay (CLEIA) Reagent for Measurement of Cyclosporine in the Blood
Authors: Tanaka M , Yui H , Baba K , Oshimo A , Hara A , Tanaka M , Ishii Y , Kaneko A , Aoyagi K
Published: October 2024

Background

Cyclosporine (CsA) is an immunosuppressive drug requiring therapeutic drug monitoring (TDM) for its narrow-ranged medication level. Approximately 60% of CsA in the blood exist as molecular complexes with proteins like cyclophilin in lymphocytes and erythrocytes. Measurement of CsA in the blood by immunoassay or liquid chromatography-tandem mass spectrometry (LC-MS/MS) requires an extraction process for CsA from the whole blood specimen, which is often manual complicated and time-consuming process causing a risk of variation in the measurement values among different operators. In addition, the small (cyclic polypeptide composed of 11 amino acids) and hydrophobic property of CsA, make it difficult to establish antibodies for a sandwich immunoassay. In this study, we have validated a novel fully automated sandwich immunoassay reagent for measurement of blood CsA that used a combination of newly-established CsA antibodies and iTACT® (immunoassay for total antigen including complex via pretreatment) technology.

Methods

The reagent was developed and validated on a fully automated immunoassay system, LUMIPULSE® L2400 (FUJIREBIO INC). Twenty uL whole blood specimens are sampled after mixed with suction/discharge stirring to prevent that blood cells settle to the bottom of a container on the analyzer, and then treated with a pretreatment solution to obtain free CsA. The treated samples are assayed by a two-step sandwich immunoassay. Limit of Detection (LoD), Limit of Quantitation (LoQ), within-laboratory precision, linearity, no interference, and the correlation of this assay and LC-MS/MS were validated, following the current CLSI guidelines protocols. Data were analyzed using Microsoft Excel statistical tool Analyse-it.

Results

The LoD and the LoQ were 2.7 ng/mL and 10.8 ng/mL respectively. The within-laboratory precision showed CV 1.6-2.9 %. Linearity was demonstrated over the range 13.1-2381.5 ng/mL. No interference was observed with unconjugated (≤19.7 mg/dL) or conjugated bilirubin (≤20.1 mg/dL), chyle (≤1480 FTU), RF (rheumatoid factor, ≤1000 IU/mL), and HAMA (human anti-mouse antibody, ≤1010 ng/mL). The correlation coefficient and the regression slope of this assay and LC-MS/MS were 1.0 and 1.0, respectively (N=120).

Conclusions

The performance of our novel CsA assay, which use LUMIPULSE L2400, was satisfactory for routine analysis. A unique fully-automated process, including the pretreatment process of whole blood specimens, for measurement would contribute the low LoQ and the low Within-laboratory CV. In addition, the results are obtained in 35-minute. We expect this novel immunoassay could reduce the burden on operators and turnaround time.
B-269 Development and CYP3A4 Characterization of a Population-based Urinary Buprenorphine and Norbuprenorphine Nomogram
Authors: Twum K , Deharvengt S J , Khan W A , Tsongalis G J , Korpi-Steiner N
Published: October 2024

Background

Buprenorphine (Bup), routinely prescribed for opioid use disorder, metabolizes to norbuprenorphine (NBup) primarily via cytochrome P450 (CYP) 3A4. Confirmation of urinary Bup and NBup helps monitor patient adherence to therapy and potential misuse. This study aims to develop and characterize a population-based urinary NBup/Bup nomogram using pharmacogenetics (PGx) to facilitate opioid result interpretations.

Methods

Adult urinary Bup and NBup results between 09/01/2022 and 09/30/2023 at UNC Health were retrieved from the EMR. Results outside the clinically reportable range (CRR; 5-25,000 ng/mL) were assigned the lower or upper CRR value, respectively. A scatterplot of Bup versus NBup/Bup on logarithmic scale was generated using Excel. Buffy coats from 14 EDTA whole blood samples were extracted and isolated using EasySep buffer (StemCell) with centrifugation at 8000rpm for 10 minutes without braking and stored at -80°C until shipment to DHMC. Genomic DNA was extracted using an EZ1 DNA procedure (Qiagen) and libraries (Agilent SureSelect) were prepared by hybridization capture of a custom PGx panel. Paired-end sequencing was performed on a MiSeq prior to diplotype-metabolizer function assignments in AUGMET. 22 CYP3A4 gene hotspots were detected by NGS and analyzed for gene-drug-variant relationships.

Results

3593 results from 896 patients were retrieved. The NBup/Bup nomogram revealed 3 subgroups from this population (Figure). CYP3A4 genotyping showed 13 wildtype calls and 1 CYP3A4 *1/*22 star allele genotype, with reduced enzyme activity. Wildtype results corresponded with Bup/NBup in subgroups 1(n=1), 2 (n=11) and 3 (n=1); CYP3A4 *1/*22 result corresponded with subgroup 3.

Conclusions

We developed a novel NBup/Bup nomogram revealing 3 subgroups in our patient population. Overlap of wildtype CYP3A4 in subgroups 1, 2 and 3 as well as CYP3A4 *1/*22 genotype in subgroup 3 suggest the NBup/Bup subgroups are independent of CYP3A4 phenotype. The characterization of population-based urinary NBup/Bup nomogram shows promise for aiding in opioid result interpretations.
B-274 Determining Novel Biomarker Candidates Associated With Heroin and Cocaine Exposure Using Urine Metabolomics
Authors: Tamama K , Patel S , Vanderschelden R A
Published: October 2024

Background

Substance abuse poses an escalating health risk in the United States. While acute drug exposure can be tracked using drugs and their metabolites as biomarkers in clinical settings, there is a notable absence of biomarkers for chronic drug exposure or addiction. The development and availability of such biomarkers could significantly enhance clinicians’ ability to identify patients at high risk of addiction, and more accurately predict disease progression and treatment responses.

Methods

Our study aimed to discover novel biomarkers associated with substance abuse by analyzing raw mass spectrometry (MS) data from urine comprehensive drug screening (UCDS) conducted at the University of Pittsburgh Medical Center (UPMC). The MS data, acquired through untargeted data collection using LC-qToF-MS at the UPMC Clinical Toxicology Laboratory, was initially preprocessed (including peak identification, alignment, and putative annotation) using MS-DIAL to generate a data matrix. This matrix, consisting of objects (cases) and features (m/z and retention time), was further normalized and analyzed using MetaboAnalyst. A combination of statistical methods, including point biserial correlation analysis, volcano plot, significance analysis of microarrays and metabolites (SAM), empirical Bayesian analysis of microarrays and metabolites (EBAM), partial least squares discriminant analysis (PLS-DA), and random forest (RF), were employed to select features significantly associated with the EIA-results. These features were subsequently evaluated for analyte identification using MS-FINDER.

Results

Our analysis identified nine significant features associated with 6MAM-EIA for heroin abuse, including 6-monoacetylmorphine itself and norfentanyl. For features associated with cocaine metabolite-EIA, 37 significant features were selected, including multiple cocaine metabolites, nicotine metabolites, and norfentanyl. Some of these selected features are likely products of in-source fragmentation or endogenous metabolites.

Conclusions

Our study has identified a set of potential biomarker candidates for illicit drug exposure. Notably, norfentanyl was found to be significantly associated with both 6MAM-EIA and cocaine metabolite-EIA, reflecting current trends in illicit drug use. Further chemical identification of these biomarkers is planned for future work.
B-275 Trends of urine drug testing findings in a pediatric hospital from 2019 to 2023
Authors: Narasimhan M , Jones P M , Poling A , Jones V , Fisher S , Forssell D , Duong T , Muthukumar A , Hashim I , Cao J
Published: October 2024

Background

Semi-quantitative immunoassays (IA) are commonly used as the initial urine drug screen (UDS), with positive results confirmed by additional testing. However, substantial false negative (FN) rates have been reported in the classes of opioids and amphetamines. In our pediatric hospital, a semi-quantitative comprehensive urine drug testing by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF) MS (referred as QTOF) is offered as either a direct order or a reflexive order following positive IA UDS. The lab also reflexes to run an IA UDS with every direct QTOF order. This study presents paired urine drug IA and QTOF results in the classes of opiate, synthetic opioid, and amphetamine/methamphetamine between 2019 and 2023 to understand trends and to evaluate assay accuracy.

Methods

Paired results from IA (Siemens Atellica) and QTOF (Sciex 4600) were analyzed with reporting cutoff concentrations from representative drugs in each class listed in Figure 1. The opiate class reported on QTOF includes 6-monoacetylmorphine, morphine, hydrocodone, codeine and metabolites. The synthetic opioid class includes buprenorphine, fentanyl, levorphanol, methadone, naloxone, oxycodone, tramadol and metabolites. Simultaneous findings of opiate and synthetic opioid were counted as the opiate class. FN rate of IA was calculated using QTOF as the reference.

Results

As shown in Figure 1, QTOF testing nearly doubled from 2019 to 2023, with an expected high FN rate in the synthetic opioid class and alarmingly rising FN rates in opiate and amphetamine classes. Within the synthetic opioid class, percentage of positive fentanyl results increased from 37.6% to 59.0% in 5 years, and ∼10% are only positive for norfentanyl, which is detected in very few commercial fentanyl IAs.

Conclusions

Our study clearly demonstrates the gap in pediatric toxicology investigation, reporting high FN rates in common drug classes by urine IA, calling for more comprehensive and sensitive UDS to be broadly adopted.
B-278 Evaluation of serum copper, zinc and selenium reference intervals using The National Health and Nutrition Examination Survey (NHANES) data
Authors: Boyd J , Yu L , Burkhead J
Published: October 2024

Background

Serum copper, zinc and selenium, are commonly ordered for assessment of nutrient status or investigation of deficiency or overload. Review of reference intervals for these analytes is challenging as most laboratories do not have the resources to do a de novo reference interval study and many literature references are not recent. The National Health and Nutrition Examination Survey (NHANES) survey presents a unique opportunity to access a comprehensive database that includes biochemical parameters and health information in order to review reference intervals regularly, especially for more esoteric tests such as trace elements. In this study, copper, zinc and selenium reference intervals were calculated using NHANES data.

Methods

NHANES data for individuals 18 years of age and older was obtained from three releases between 2012-2016. Copper, selenium and zinc 95% confidence intervals were evaluated by age, sex and body mass index (BMI) partitions using quantile regression. All calculations were performed in R using the quantreg package.

Results

The calculated reference intervals for serum copper, zinc and selenium are shown in Table 1. Serum copper had the largest changes observed between sexes, with females having a much wider reference interval on the high end. Oral contraceptive use is known to increase serum copper levels which likely explains this trend. BMI over 30 also increased the serum copper reference interval, more clearly in women than in men. Zinc reference intervals were more aligned between women and men, with slightly higher intervals observed in men. BMI did not have much effect in either sex. For selenium, men had slightly higher reference intervals than women.

Conclusions

NHANES data provides a comprehensive data set for reference interval evaluation, especially for more esoteric analytes such as trace elements.
B-292 Detection of natural toxins derived from potatoes by biotin-streptavidin direct ELISA
Authors: Okada K
Published: October 2024

Background

α-solanine (SO) and α-chaconine (CHA) are natural toxins of potato and often cause food poisoning worldwide. We have previously developed a polyclonal antibody (anti-Sold antibody) that bind to SO and CHA and used it in an enzyme-linked immunosorbent assay (ELISA). Our previously constructed ELISA (conventional ELISA) has a weak point of low sensitivity in detecting SO and CHA, we have tried to improve it in this study.

Methods

Solanidine, a chemical compound found in both SO and CHA, was conjugated with bovine albumin, and this complex was repeatedly injected into two rabbits for two months. Anti-Sold antibody was then purified from rabbit serum using affinity column chromatography. In our ELISA method, named “B-S direct ELISA”, biotin-labeled anti-Sold antibody is bound to SO and CHA in the sample, followed by the binding of peroxidase-labeled streptavidin. To simulate biological samples from patients with potato food poisoning, SO and CHA powders were mixed with commercially available human serum and urine, and SO and CHA levels were then measured using ELISA. Lastly, SO and CHA levels were detected in extracts from rotten potato (Irish Cobbler) sprouts, peels, and tubers.

Results

The absorbance of the samples containing SO and CHA prepared in 10 mM phosphate buffer saline (PBS) was about 5-fold higher than that of the conventional ELISA. Furthermore, the absorbance of the samples containing SO and CHA prepared in human serum and urine was enhanced about 2.5- and 1.6-fold, respectively, compared to that of the conventional ELISA. The limit of detection in B-S direct ELISA was 0.32 ng/mL in 10 mM PBS, 2.08 ng/mL in serum, and 4.88 ng/mL in urine, respectively. Using this ELISA, it was also possible to measure the content of SO and CHA in extracts of potato tubers, peels, and sprouts. Both SO and CHA in the extracts from rotten potato tubers and peels were detected as approximately 1.4- and 2.2-fold higher, respectively, in each part than those in the extracts from fresh potatoes. Furthermore, the potato sprout extract contained approximately 50-fold more SO and CHA than the tuber and 9-fold more than the peel. On the other hand, B-S direct ELISA cross-reacted with solanidine and solasodine, which have similar chemical structures to SO and CHA, and also with cholesterol and vitamin D3 in serum.

Conclusions

Based on our results, B-S direct ELISA presented greater performance in detecting SO and CHA than our conventional ELISA. The non-specific reactions in the serum samples and influence of urea in the urine samples may underlie the reduced detection performance of SO and CHA in ELISA, respectively. Although this ELISA has a detection performance applicable to human biological samples, cross-reactivity should be fully considered when judging the results.
B-294 Case report: Postmortem ethanol production following traumatic injury
Authors: Whitt A , Alquran B , Jortani S A
Published: October 2024

Background

Ethanol is the most commonly encountered psychoactive substance in forensic toxicology. Accurate interpretation of ethanol concentrations from postmortem samples is fraught with potential hurdles, including decomposition, redistribution, and postmortem production of ethanol. Through the fermentation of substrates like glucose, pyruvate, lactate, and amino acids, microorganisms present in the gut and/or environment can generate ethanol in contaminated human tissues, especially following traumatic injury. The degree of fermentation varies widely depending on the time between death and autopsy, temperature, humidity, tissue glucose concentrations, and the specimen type. Vitreous humor (VH) is the preferred specimen for postmortem toxicological analysis of ethanol because it accurately reflects the antemortem ethanol concentration in serum while being resistant to microbial contamination. This case highlights the utility of VH in an extreme instance of postmortem ethanol production.

Methods

Postmortem toxicological analysis of liver tissue and VH (NMS Labs, Horsham, PA) was performed by headspace-gas chromatography (HS-GC)

Results

Case presentation: A man in his sixties with a history of type 1 diabetes mellitus and hypertension was involved in a fatal mining accident while operating heavy machinery in which the equipment fell down a cliff, causing the man to be ejected onto the ground nearby. The decedent was discovered hours after the fall with extensive traumatic injuries to the left side of his body and head. Seven days later, an autopsy was performed by the medical examiner’s office. Postmortem toxicological analysis of liver tissue by HS-GC revealed ethanol at a concentration of 690 mg/100g. The liver tissue also tested positive for amlodipine, nicotine, cotinine, and caffeine. A vitreous humor (VH) sample tested negative (<10 mg/dL) for ethanol.

Conclusions

The toxicological findings reported herein are inconsistent with antemortem alcohol consumption and are instead an example of severe postmortem ethanol production by microbial contamination. Several factors likely led to the stark discrepancy between the concentrations of ethanol in VH and liver tissue. First, the decedent suffered traumatic injuries, creating conditions favorable for extensive microbial contamination. Second, the time between death and autopsy was seven days, allowing ample time for fermentation to occur, even under refrigerated conditions. Third, postmortem glycogenolysis in addition to the patient’s underlying T1DM likely provided a high concentration of glucose to fuel postmortem fermentation. Ultimately, this case underscores the importance of forensic specimen selection in accurately interpreting toxicology findings following traumatic injury.
B-303 Stability of Antipsychotic Drugs in Patient Serum and Plasma Clinical Samples
Authors: Konrath M S , Drisgill A , Kozo D , Hilaire M , Salamone S J
Published: October 2024

Background

Therapeutic drug monitoring (TDM) of antipsychotic blood levels is strongly recommended (clozapine and olanzapine) and recommended (risperidone, aripiprazole, and quetiapine) by ASCP/AGNP Consensus Statement. For effective TDM, it is paramount to understand both the stability of the antipsychotic drugs in serum and plasma, as well as any differences in levels due to sample matrix to ensure robust concentration determinations. While clozapine levels are considered equivalent in serum and plasma, we included in our study four additional antipsychotics.

Methods

Whole blood samples from ten patients taking clozapine, risperidone, aripiprazole, olanzapine, or quetiapine were collected in one red top tube and in one purple top tube according to an IRB-approved protocol. Samples were then processed within 2 hours of collection to obtain matched serum and K2EDTA plasma samples from each of the patients. CE marked immunoassays for the measurement of clozapine (also FDA cleared), total risperidone/paliperidone, total aripiprazole, olanzapine, and quetiapine were used to quantitate drug concentrations. Stability of the samples was assessed at 4°C, ambient room temperature (ART), and frozen at -80°C, with respect to their Day 0 concentration by measurement of six replicates at each timepoint using the immunoassays.

Results

Comparison of Day 0 values between serum and plasma for the same patient showed ±9% bias between sample matrices for all drugs, except olanzapine which showed differences up to ±15%. Average bias between serum and plasma was ≤-5% (olanzapine: -9%). All drugs were stable (±15% deviation from Day 0; olanzapine up to ±23%) in serum and in plasma for 7 days at both 4°C and ART and for up to 8 months frozen.

Conclusions

This study demonstrated that (1) the antipsychotic drugs clozapine, risperidone, aripiprazole, olanzapine, and quetiapine have sufficient stability in both serum and plasma for effective TDM and (2) for TDM, serum or plasma may be used.
B-349 Diagnostic Performance of the Atellica IM High-Sensitivity PSA Assay in Men Undergoing Prostate Cancer Screening
Authors: Olson Nissen K , Snyder J , Pagano M , Owusu A , Grosett M , Birmingham N , Christenson R , deFilippi C
Published: October 2024

Background

This study evaluated the diagnostic performance of the Atellica® IM High-Sensitivity PSA (hsPSA)* assay in men >50 years undergoing prostate cancer (PC) screening in conjunction with digital rectal exam (DRE).

Methods

Serum samples were collected prospectively from 2671 subjects >50 years referred to a urologist for evaluation of PC from 26 sites across the United States. To establish PC versus benign disease, all subjects underwent a transrectal prostate biopsy ≤60 days after blood draw. At the cutoff of 4.0 ng/mL for the hsPSA assay, the percentage of PCs detected (sensitivity) and corresponding percentage of biopsies avoided in men without PC (specificity) were determined.

Results

Cancer prevalence (malignant biopsy) was 58.5% (1562/2671) among the diagnostic population. Preliminary results demonstrated that for DRE alone, sensitivity and specificity were 30.9% (482/1562) and 83.0% (920/1109) compared to biopsy. For hsPSA alone at the cutoff of 4.0 ng/mL, sensitivity was 88.7% (1386/1562). The table provides incidence of cancer and positive biopsies by subgroups defined by DRE and hsPSA. hsPSA detected 61.1% (955/1562) of PCs that DRE did not. Of those DRE+, 71.8% (482/671) had positive biopsy. Of those DRE+ and hsPSA ≥4.0 ng/mL, 78.6% (431/548) had positive biopsy, which significantly increased PC detection by 7.0% (95% CI:1.4%-12.5%). Similarly, in the DRE- population, while 54.0% of subjects had positive biopsy, subjects both DRE- and hsPSA <4.0 ng/mL had only 38.5% positive biopsies, a significant difference of 16.5% (95% CI: 6.3%-27.8%).

Conclusions

Using DRE alone detected only 3 out of 10 patients with PC. It was observed that when used in conjunction with DRE, the hsPSA assay improved both sensitivity and specificity of PC detection over DRE alone.

CAUTION: Investigational Device. Not available for sale. Future availability cannot be guaranteed. The results are preliminary and were achieved in unique setting with no expected timeline for completion.
B-352 Effects of COVID-19 on Breast Cancer Positivity: A Retrospective and Predictive Study
Authors: de Andrade S S , Moreno Rodrigues de Souza G , Pagan Cilotti H , Ribeiro Ramadan-Boscolo D , Tufik S
Published: October 2024

Background

The COVID-19 pandemic has had a significant impact on the healthcare system, resulting in overall reduction in access to healthcare services, including the suspension of screening programs and postponement of surgeries and medical treatments. The aim of this study was to evaluate the effects of the pandemic on breast cancer positivity from cases received at a laboratory in São Paulo, Brazil.

Methods

A retrospective analysis of a database from a pathology laboratory in São Paulo, Brazil, was performed during the 2017-2023 period and classified as following: pre-pandemic (January 2017-February 2020), pandemic (March 2020 - April-2022), and post-pandemic (May 2022-December 2023). Cases were classified as negative or positive for malignancies. Positive anatomopathological examinations (breast biopsies and surgical specimens) considered primary, metastatic mesenchymal or endothelial malignancies. The Mann-Kendall test was used to evaluate the trend, and the PACF (Partial Autocorrelation Function) correlogram was used to analyze historical memory for definition of the autoregressive model. The augmented Dickey-Fuller hypothesis test was applied to analyze whether the series is stationary or not, and the first differences test was applied. The ARIMA model was used for the estimates (1,1,1). Statistics were performed using the gretl software and pymannkendall library in the Python language.

Results

A total of 74,973 samples were evaluated during the 2017-2023 period. In the three-year pre-pandemic period, 32,644 tests were recorded, with an average of 29.34% positive cases. From the analysis of the pre-pandemic time series, there was a positive trend towards an increase in cases (p < 0.0001, z=6.1360, Tau=0.6955). In a non-pandemic scenario, from March 2020 to February 2021, the number of new tests expected would be 11,535; however, in the pandemic period, only 8,904 were observed, a 22.81% reduction on expected cases. According to the historical average positivity rate in the pre-pandemic period, the expected and observed total were, respectively, 3,384 and 2,566 positive cases for malignancy.

Conclusions

This study reveals a significant decrease of approximately 22.81% in new breast anatomopathological diagnoses during the COVID-19 pandemic in a laboratory in São Paulo, Brazil. For comparison purposes, we identified a 24.17% of missed opportunities on diagnoses of new malignancy-positive cases, compared to the pre-pandemic period. These findings highlight the need for strategies to mitigate the negative effects of the pandemic on the early detection and treatment of breast cancer.
Two leucine-rich repeat receptor-like kinases initiate herbivory defense responses in tea plants
Authors: Jiang Qi , Ding Changqing , Feng Lingjia , Wu Zhenwei , Liu Yujie , He Lintong , Liu Chuande , Wang Lu , Zeng Jianming , Huang Jianyan , Ye Meng
Published: October 2024

Leucine-rich repeat receptor-like kinases (LRR-RLKs) have emerged as key regulators of herbivory perception and subsequent defense initiation. While their functions in grass plants have been gradually elucidated, the roles of herbivory-related LRR-RLKs in woody plants remain largely unknown. In this study, we mined the genomic and transcriptomic data of tea plants (Camellia sinensis) and identified a total of 307 CsLRR-RLK members. Phylogenetic analysis grouped these CsLRR-RLKs into 14 subgroups along with their Arabidopsis homologs. Gene structure and conserved domain analyses revealed notable similarities among subgroup members. Among the identified CsLRR-RLKs, we focused on two plasma membrane-localized LRR-RLKs, CsLRR-RLK44, and CsLRR-RLK239, which do not form homodimers or heterodimers with each other. Both respond strongly to herbivory, and their expression patterns significantly correlate with herbivore resistance phenotypes across different tea accessions. CsLRR-RLK44 and CsLRR-RLK239 act upstream of mitogen-activated protein kinase (MPK) cascades and modulate the expression of defense-related MPKs and WRKY transcription factors. Additionally, silencing CsLRR-RLK44 or CsLRR-RLK239 reduced the levels of herbivory-induced jasmonates, thereby weakening the plant resistance to tea geometrid larvae (Ectropis obliqua). Our work is the first to demonstrate that in woody plants, LRR-RLKs are essential for enhancing herbivore resistance through the activation of the canonical signaling, including MPKs, WRKYs, and jasmonates. Furthermore, our study extends mechanistic insights into how LRR-RLKs initiate plant defenses from grasses to economically important tree species.
SlSTOP1-regulated SlHAK5 expression confers Al tolerance in tomato by facilitating citrate secretion from roots
Authors: Zhu Huihui , Chen Weiwei , Yang Zheng’an , Zeng Congfang , Fan Wei , Yang Jianli
Published: October 2024

SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) is a core transcription factor that regulates the expression of aluminum (Al) resistance genes to manage Al toxicity in plants. However, the genome-wide roles of SlSTOP1 in the Al stress response of tomato (Solanum lycopersicum) remain largely unknown. Here, we report that SlSTOP1 is crucial for Al tolerance in tomato, as loss-of-function mutants of SlSTOP1 displayed hypersensitivity to Al stress. Aluminum stress had no effect on SlSTOP1 mRNA expression, but promoted accumulation of SlSTOP1 protein in the nucleus. Through integrated DNA affinity purification sequencing and RNA sequencing analysis, we identified 39 SlSTOP1-targeted Al-responsive genes, some of which are homologous to known Al resistance genes in other plant species, suggesting that these SlSTOP1-targeted genes play essential roles in Al resistance in tomato. Furthermore, using peak enrichment analysis of SlSTOP1-targeted sequences, we identified a cis-acting element bound by SlSTOP1 and validated this finding via dual-luciferase reporter and electrophoretic mobility shift assay (EMSA). Additionally, we demonstrated SlHAK5 is one of direct targets of SlSTOP1 and functionally characterized it in terms of Al stress tolerance. Compared with wild-type plants, Slhak5 mutants developed by CRISPR/Cas9 technology presented increased sensitivity to Al stress, which was associated with reduced citrate secretion from the roots. Together, our findings demonstrate that SlSTOP1 directly interacts with cis-acting elements located in the promoters of target genes involved in diverse pathways contributing to Al resistance in tomato.
Synovial sarcoma of the ethmoid sinus with extension to the cavernous sinus: a case report and literature review
Authors: Sharabati Haneen H , Innab Laila R , Hussein Saja S , Salman Ayman A , Naser Anis M , Bakri Izzeddin A
Published: October 2024

Synovial sarcomas are rare soft tissue tumors primarily affecting the extremities but can occasionally manifest in unusual locations such as the ethmoid sinus, posing diagnostic challenges. We present a case of a 38-year-old male with a 7-month history of recurrent throbbing headaches, left eye pain, and facial nerve palsy, evolving into multiple stroke episodes. Radiological studies showed extension to the cavernous sinus, raising an initial suspicion of vasculitis. Histological findings of an endoscopic biopsy, however, confirmed a monophasic synovial sarcoma. The patient was referred to a specialized center for further management. Unfortunately, he developed another stroke before receiving treatment. Management included chemotherapy and definitive radiation therapy targeting the ethmoid sinus. The patient is currently receiving ongoing palliative care for symptom management. This case underscores the importance of early diagnosis and a multidisciplinary approach in managing rare and aggressive tumors such as synovial sarcoma of the ethmoid sinus.
Revisiting the early light-induced protein hypothesis in the sustained thermal dissipation mechanism in yew leaves
Authors: Ye Zihao , Sawada Mina , Iwasa Makiko , Moriyama Ryo , Dey Debayan , Furutani Miyu , Kitao Mitsutoshi , Hara Toshihiko , Tanaka Ayumi , Kishimoto Junko , Yokono Makio , Akimoto Seiji , Takabayashi Atsushi , Tanaka Ryouichi
Published: October 2024

Overwintering evergreen trees in boreal regions continuously convert absorbed light energy into heat through a process known as sustained thermal dissipation. To better understand this mechanism, this study examined the alterations in the photosynthetic apparatus and transcriptomes of yew (Taxus cuspidata) leaves throughout the year, comparing sun-exposed and shaded leaves. The Y(II) parameter, conventionally used to estimate the quantum yield of photosystem II (PSII), indicated the occurrence of temperature-dependent thermal dissipation during winter. On the other hand, the levels of photosystem subunits, including the D1 subunit of the PSII reaction center, remained relatively stable year-round, indicating that typical photoinhibition is unlikely to occur. Time-resolved chlorophyll fluorescence analysis revealed that heat dissipation at the PSII antenna is prominent in winter. Winter transcriptomes are notably characterized by a predominance of Elip transcripts encoding early light-induced protein (ELIP), which constitute 20% of the total transcripts, as deduced from RNA-seq analysis. Furthermore, ELIP protein concentration increased to nearly half that of the major light-harvesting complexes. The predicted structure of ELIP includes potential chlorophyll a and carotenoid binding sites. These findings, taken together with a previous report showing ELIP capacity for energy dissipation, lead to a re-evaluation of its significant role in sustained thermal dissipation.
Alleviating the Hubble tension using the Barrow holographic dark energy cosmology with Granda–Oliveros IR cut-off
Authors: Yarahmadi M , Salehi A
Published: October 2024

In this study, we investigate the Barrow holographic dark energy (BHDE) model with the Granda–Oliveros(G–O) infrared (IR) cut-off in the presence of neutrino masses, utilizing the latest observational data to address the Hubble tension. The GO cut-off is defined as $L_{\mathrm{ IR}}=(\alpha H^2 + \beta \dot{H})^{-1/2}$. We place constraints on the total neutrino mass $\sum m_{\nu }$ using data from Type Ia supernovae (SN) Pantheon, cosmic chronometers (CC), cosmic microwave background (CMB), Baryon Acoustic Oscillation (BAO) data sets, and Planck Lensing. Specifically, the comprehensive CMB + BAO + CC + Pantheon data set provides a total neutrino mass of $0.118\, \text{eV}$. The parameters for the Barrow-GO model are determined to be $\Delta = 0.0055^{+0.0086}_{-0.0086}$, $\alpha = 0.997^{+0.060}_{-0.060}$, and $\beta = 0.598^{+0.080}_{-0.080}$, showing good agreement with previous studies. One of the key findings of this study is the model’s ability to alleviate the Hubble tension, as evidenced by the comparison of $H_0$ measurements. Specifically, the tension value for the combination of data set (CMB + BAO + CC + Pantheon + Lensing) is $1.5\sigma$ with the Planck 2018 and $1.4\sigma$ with R22. These results underscore the importance of multi-data set integration in refining constraints on neutrino properties and highlight the model’s efficacy in probing fundamental aspects of neutrino physics. Our results demonstrate that the BHDE model with the GO cut-off can effectively address the Hubble tension, offering a coherent framework that reconciles local and cosmological measurements of the Hubble constant.