bluuebunny/crossref_metadata_2025_split huggingface dataset:
B-225 Assessment of the Genotype Frequency of Thiopurine Methyltransferase (TPMT) Deficiency in a Large Cohort of Patients With Immune Mediated Inflammatory Disease and Cancer
Authors: Stachelski J , Pham D , Hughes P , McLachlan O , Dervieux T 
Published: September 2023

Abstract

Background
Thiopurine methyltransferase (TPMT) alleles (*1/*2/*3A/*3B/*3C) are routinely genotyped to determine the metabolizer status of patients that plan to undergo thiopurine therapies (6-Mercaptopurine and Azathioprine). Screening of these alleles allows for personalized patient dosing and reduces the risk of severe or even fatal myelosuppression. The objective of this study was to determine the genotype frequency of TPMT deficiency in a large clinical cohort of patients with immune mediated inflammatory diseases and cancer.

Methods
All genotyping was performed in a reference clinical laboratory. Patient specimens were tested using genomic DNA isolated from whole blood. Clinical patient diagnoses were physician provided (international classification of disease, ICD-10). The genomic DNA was tested for the presence of three single nucleotide polymorphisms (SNPs) in the TPMT gene coding region and known to associate with reduced TPMT activity (rs1800462-Exon 5, rs1800460-Exon 7 and rs1142345-Exon 10). These polymorphisms determine the presence of *2/*3A/*3B/*3C alleles. Testing occurred from 10/26/2000 to 9/9/2022. Three different testing technologies were used during the 22 years of testing (PCR-RFLP from 2000 to 2003, Taqman™ chemistries from 2004 to 2015, and Luminex™ xMAP™ bead-based since 2016).

Results
A total of 219 129 patients were tested (mean age: 38 yrs, 56% female). As presented in the Table the genotype frequency for the wild type *1/*1 was 90.8% (95%CI: 90.7–90.9%), it was 9.0% (95%CI: 8.8–9.1%) for the heterozygous and 0.22% (95%CI: 0.020–0.024%) for homozygous variant, with similar frequency across disease states. The TPMT *1/*3B genotype frequency was very rare (&lt;0.1%, n = 5) and the *3B/*3C homozygous variant (which cannot be distinguished from the *1/*3A) was calculated to have a frequency of 1:7 706 230 (95%CI: 1:7 043 228–1:8 369 232) and lower than initial estimates.

Conclusion
We estimated the frequency of TPMT genotypes in the largest cohort reported to date. The occurrence of the *3B/*3C genotype may be rarer than initially estimated.
B-246 The Evaluation of Actionable Mutations By Multiplex Analysis Using IntelliPlex® Lung Cancer Panel for Non-Small Cell Cancer Management
Authors: Cheng W 
Published: September 2023

Abstract

Background
Understanding the genetic alterations which occur in genes associated with various cancers can improve therapy outcomes by promoting tailored treatments for each patient. Molecular profiling is therefore a crucial tool for precision medicine and enables for a personalized approach to cancer management. Current guidelines for molecular testing of Non-Small Cell Lung Cancer (NSCLC) patients involves treatment selection of a targeted Tyrosine Kinase Inhibitor based on the minimal testing of EGFR, ALK and ROS1 genes and strongly recommending the additional testing of KRAS, BRAF, MET, RET, NTRK and ERBB2 (Her2) genes to fully evaluate the patient’s mutational status.

Method
The IntelliPlex Lung Cancer Panel, based on PlexBio’s multiplexing πCode® technology, is an in vitro molecular amplification assay intended for the qualitative identification of 74 DNA mutations in the KRAS, NRAS, PIK3CA, BRAF, EGFR, HER2, MEK1 and AKT1 genes and 28 gene variants of the ALK, ROS1, RET, NTRK1 and MET genes. The assay is suitable for the use with formalin-fixed paraffin-embedded (FFPE) tumor tissues from NSCLC patients. The performance of IntelliPlex Lung Cancer Panel was evaluated on FFPE specimens by comparing the results with two commercially available molecular amplification assays. Discrepant samples were resolved by a corresponding digital PCR method kit.

Results
The IntelliPlex Lung Cancer Panel can detect DNA mutations at very low allele frequencies (as low as 0.1%) while only requiring DNA quantities of 10ng. Most RNA rearrangements are detected with 10 or more copies present in the sample while only low RNA input quantities (50 ng) are required. The IntelliPlex Lung Cancer Panel can analyze the mutational status of 102 targets in less than 5.5 h from sample to results and allows screening of up to 48 samples in parallel. Among 50 FFPE DNA specimens tested, the overall agreement for EGFR and KRAS mutations between IntelliPlex system and the comparison method were both found to be 98%. And the overall agreement after resolved the discrepancies by digital PCR was 100%.

Conclusion
The multiplex amplification and detection capabilities of the IntelliPlex Lung Cancer Panel provides a comprehensive and efficient way to evaluate the individual mutational status from lung cancer patients with high sensitivity and specificity. Accurate genetic analysis provides valuable information which can support precision medicine decisions for better cancer management. This can also be used further with non-invasive whole blood sampling (i.e., “liquid biopsy”) for subsequent treatment monitoring.
B-247 Rapid and Field-deployable Microchip-based Real-time PCR for Hepatitis B Virus DNA Detection
Authors: Liu Y , Gao Y , Song Q , Hong Y , Wen W 
Published: September 2023

Abstract

Background
Hepatitis B is one of the most common infectious diseases with global significance, seriously causing chronic liver complications and a heavy health burden in developing countries. There is no doubt that real-time polymerase chain reaction (RT-PCR) has been a golden standard for monitoring the Hepatitis B virus (HBV) level in clinical health management. However, the conventional PCR protocol cannot fulfill the field test requirement in some resource-poor areas due to its drawbacks, such as huge size, long turnaround time, and demand for professional laboratory facilities. For solving these problems, a portable microfluidic chip-based PCR detection system was developed to enable the nucleic acid test in a flexible, rapid, and economical way. With the efficient temperature control module and advanced optical path design, this novel panel can achieve fast thermal circulation with a 30°C/s heating rate and multiple fluorescent detection with a throughput of 6 samples at a time.

Methods
In this study, we assessed the detection performance of the system using a commercial HBV nucleic acid detection kit. The reproducibility, limit-of-detection (LoD), and linearity were validated by reference samples with gradient concentrations. We evaluated the clinical sensitivity and specificity of the microdevice by a total of 200 extracted serum samples (100 positive and 100 negative cases). The analytical results were compared with a traditional PCR device with FDA certification.

Results
The variation coefficients of the microdevice for reference samples with different concentrations are 1.78%, 1.98%, and 1.50% (n = 10), respectively. The field-deployable PCR system can provide the HBV DNA assay with a detection limit of 300 IU/ml, whose reaction time was approximately 67 mins rather than the 108 mins performed by the comparator. During the establishment of the standard curve, this fast PCR test shows a strong linear correlation (R2 = 0.9919) between targeted gene concentration and dilution factor. Additionally, among the 200 clinical samples, we used the microchip-based system to rapidly complete quantitative detection for each sample with high sensitivity of 100% (100/100) and specificity of 100% (100/100), which is critically consistent with the traditional PCR method.

Conclusion
Hence, the point-of-care microfluidic PCR system exhibits great feasibility in offering timely, accurate, and reliable molecular diagnosis for early epidemic disease screening and therapeutic guidance of chronic infections.
B-293 Non-renal Sources of Erythropoietin: Complications for Interpreting Erythropoietin Assays
Authors: Alshamali R , Marrow J P , Platt M J , Brunt K R , Simpson J A 
Published: September 2023

Abstract

Background
Erythropoietin (EPO) is a glycoprotein that stimulates the production of red blood cells, in response to systemic hypoxia. Generally, the kidney is thought to be the sole producer of EPO. Emerging data shows Epo mRNA expression can occur in non-renal tissues, yet the significance of this remains uninvestigated. Clinically, EPO assays are primarily used to identify the underlying cause of anemia and polycythemia. Interestingly, EPO serum levels are elevated following a myocardial infarct (MI); the source is always assumed to be kidney with no further investigations into non-renal sources of EPO. Here we investigate the heart as a novel source of serum EPO following a MI. We hypothesize that the heart, not the kidney, produces EPO in response to organ-specific hypoxia (i.e., MI).

Methods
To assess which organs are producing EPO following a MI, we examined EPO mRNA expression. mRNA levels correlate strongly with EPO protein synthesis, as EPO is exclusively regulated at the transcription level. To induce a MI, CD1 mice were subjected to ligation of the lateral anterior descending artery. Left ventricle and kidney tissues were collected at 24 h, 4 weeks and 18 weeks following MI or sham surgery; tissue was snap frozen and stored at −80°C for qPCR analysis. Hematocrit was measured at 2-, 4-, and 18-weeks following MI or sham surgery.

Results
24 h following a MI, the left ventricle showed a ∼100-fold increase in EPO mRNA expression in comparison to sham group (P = 0.0012); the kidney showed no significant differences in EPO mRNA between MI and sham group. Hematocrit was measured from the carotid artery. At 2- and 4- weeks following surgery, hematocrit in MI group was significantly elevated in comparison to baseline hematocrit levels (Baseline: 40.8% ± 4, 2-week: 47.8% ± 3.1, and 4-week: 46% ± 2.6 (P = 0.0003, P = 0.0103, respectively). Whereas there were no significant differences in hematocrit between baseline and the sham group.

Conclusion
Here we show that following a MI, the heart, not the kidney, produces EPO. Subsequently, there is an increase in hematocrit as a result of cardiac-specific EPO production. Further, perturbations in EPO serum levels are not disease specific. In cases where EPO assays are conducted on MI patients, interpretation of EPO levels should be cautioned as it may not be exclusively diagnostic of the underlying cause of anemia or other bone marrow disorders. Therefore, an alternative diagnosis may be warranted for elevated EPO levels in this specific patient population.
B-294 Delineating the Diversity of Recombinant Monoclonal Antibodies Isolated From Alpaca Polyclonal Mixture
Authors: Narang D , Le Bihan T , De Villavicencio Diaz T N , Ma B 
Published: September 2023

Abstract

Introduction
Polyclonal antibodies are a mixture of antibodies produced by plasma B-cells and are often obtained by injecting a specific antigen into a host system. They are commonly used for research and diagnostic purposes and are inexpensive and relatively quick to produce. But they lack batch-to-batch reproducibility making them less reliable than recombinant monoclonal antibodies (mAbs). Our team designed a polyclonal sequencing method to sequence the mAbs directly from the immunoserum and then used recombinant technology to reliably express the mAbs. The diversity of the epitopes of these recombinant mAbs are then examined with Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS).

Methods
In this study, we used a novel mass-spectrometry-based approach to do de novo sequencing of the Alpaca IgGs directly from the immunoserum without analyzing the RNA-seq data. Using our established REpAb sequencing platform and a machine learning-based bioinformatic analysis, we successfully compiled complete amino acid sequences of individual alpaca IgG clones found in the original polyclonal mixture. Using this approach, we isolated ten unique sequences from the polyclonal mixture and expressed them recombinantly. In addition, we performed Hydrogen-deuterium exchange Mass spectrometry experiments on 4 of the ten mAbs to identify their binding sites. For HDX analysis, the antigen digestion was performed using Pepsin/protease XIII column, and the peptides were identified using Waters SELECT SERIES Cyclic IMS Mass spectrometer.

Preliminary data
The four mAbs which showed strong binding to the antigen were chosen to characterize further using HDX-MS experiments. The preliminary MS/MS experiments provided us with approximately 90% of the amino acid sequence coverage of the antigen. Interestingly, the HDX-MS results showed that different mAbs bind to different antigen regions, which is evident from the deuterium exchange. Overall, our results showed that the monoclonal antibodies isolated from the polyclonal mixture bind to different antigen regions and can represent the diversity in the polyclonal mixture.

Novel aspect
De novo sequencing functional mAbs from immunoserum and studying their epitope diversities with HDX-MS may help discover novel therapeutics antibodies.
B-306 Performance Evaluation of an Automated Assay for Measurement of Tacrolimus* on the ADVIA Centaur XP System
Authors: Chuang T , Swan R , Sankiewicz O , Casson N , Baxter S 
Published: September 2023

Abstract

Background
Tacrolimus (FK506) (PROGRAF) is a macrolide antibiotic of fungal origin (Streptomyces tsukubaensis). Tacrolimus is an inhibitor of calcineurin, a phosphatase that activates T-cell proliferation. At the cellular level, tacrolimus binds a family of binding proteins termed FK506-binding proteins (FKBPs). The correlation between steady-state trough concentration and area under the curve (AUC) provides reliable therapeutic monitoring for tacrolimus exposure at the trough level. The objective of this study was to determine the performance characteristics of the Tacrolimus assay on the ADVIA Centaur® XP System.

Methods
The ADVIA Centaur Tacrolimus assay is a competitive immunoassay using direct chemiluminescent technology. Free tacrolimus in the patient sample competes with the bound tacrolimus in the solid phase for binding with the acridinium ester-labeled anti-tacrolimus antibody. The amount of tacrolimus present in the patient sample has an inverse relationship to the amount of relative light units detected by the system.

Results
The ADVIA Centaur Tacrolimus assay demonstrated a mean %CV of 3.1% for intra-assay and 6.0% for total precision (across a sample range of 3.10 to 26.15 ng/mL). Close correlation to LC-MS/MS was demonstrated, with r, slope, and intercept of 0.957, 0.973, and 0.475, respectively. LoD and LoQ were 0.7 ng/mL and 1.9 ng/mL, respectively. Likewise, comparison to the Atellica IM assay resulted in r, slope, and intercept of 0.966, 1.09, and -0.508, respectively. Linearity was demonstrated across a range from LoQ to 30.0 ng/mL. Tacrolimus metabolite percent cross-reactivity ranged from 1.7 to 34.2%. A panel of potential interferants including the following were tested and found not to interfere (≤10%): cephalosporin to 100 µg/mL, erythromycin to 13.8 mg/dL, phenobarbital to 10 mg/dL, rapamycin to 5 µg/mL, sulfamethoxazole to 150 µg/mL, tobramycin to 3.3 mg/dL, trimethoprim to 4.2 mg/dL, bilirubin (conjugated) to 60 mg/dL, bilirubin (direct) to 60 mg/dL, and rheumatoid factor to 500 IU/mL.

Conclusion
The resulting data demonstrates that the ADVIA Centaur Tacrolimus assay is an accurate and precise method of quantifying tacrolimus concentration in EDTA whole-blood samples. *Assay under development by Randox Laboratories Ltd. for Siemens Healthineers. Not available for sale. Future availability cannot be guaranteed.
B-323 Urinary Thebaine Improves Opioid Interpretation in Pain Management
Authors: Pourfarrokh N , Nguyen N 
Published: September 2023

Abstract

Background
Poppy seed from Papaver somniferum plant is used in bakery results in positive urine test for Morphine and Codeine up to 48 h. This leads to prescription compliance failure in the setting of clinical pain management. To mitigate misinterpretation, Thebaine testing was introduced to confirmatory opioid panel. Thebaine is naturally found in poppy seeds but not in prescription opiate formularies. A positive Thebaine result helps identify poppy seed consumption rather than from opioid prescription abuse.

Method
Each of the seven healthy adult volunteers (age 20–50) consumed one poppy seed filled kolache. Urine samples were then collected from each person at pre and post-consumed time points at 2, 4, and 24 h. All samples were screened on Abbott Architect c16000 immunoassay (Abbott Park, IL) for opioids at 300 ng/mL cutoff. The samples were quantitated using ThermoFisher Vanquish Quantis LC-MS/MS (Waltham, MA) for Codeine, Morphine, and Thebaine at AMR 10–500 ng/mL, 10–500 ng/mL, and 5–250 ng/mL, respectively. Data acquisition/analyses were performed on TraceFinder software. The deuterated internal standards were purchased from Cerilliant Corporation (Round Rock, TX). All samples met calibrators and quality controls quality criteria.

Results
All post-consumption immunoassay results were positive for opioid. All LC-MS/MS positive results showed various levels of Codeine, Morphine, and Thebaine at 230–4300 ng/mL, 9–290 ng/mL, and 5–210 ng/mL, respectively (Fig. 1).Fig. 1.

Conclusion
Poppy seed consumption results in positive opioid class by immunoassay and positives Codeine, Morphine by LC-MS/MS. Detection of Thebaine in these samples would enable accurate clinical interpretation for poppy seed dieters and abusers of Codeine and Morphine prescription.
B-325 Retrospective Study Comparing Immunoassay Screen and Mass Spectrometry Confirmation Results in an Opioid Dependant Population
Authors: Bennett M J , Luong J , Kinniburgh D W 
Published: September 2023

Abstract

Background
Retrospective analysis of clinical laboratory data is employed to monitor the performance of instruments, evaluate test utilization and support operational changes. Urine drug testing in the context of opioid use disorder is used to assist in the management of patients. This testing is typically performed by an immunoassay screen followed by mass spectrometry confirmation. Immunoassay screens identify drug class and can cross-react with off-target substances to produce false-positive or false-negative results. Mass spectrometry confirmations provide more sensitive and specific results. The objective of this study was to utilize historical data to determine the concordance between immunoassay and mass spectrometry results from patients in an opioid dependency treatment program.

Methods
Urine drug screening results spanning a 3-year period (2020–2023) were mined to identify samples that screened positive for amphetamines, benzodiazepines, opiates, fentanyl/carfentanil, oxycodone/oxymorphone, and cocaine. These results were compared to the mass spectrometry confirmation results. The immunoassay results were collected on an Olympus AU480 instrument and the mass spectrometry results were collected using an in-house developed dynamic multiple reaction monitoring method on both Agilent 6470 and 6460 triple quadrupole instruments.

Results
Of the 15 850 urine drug records reviewed, 28.8% screened positive for amphetamines, 23.5% screened positive for benzodiazepines, 14.2% screened positive for opiates, 23.8% screened positive for fentanyl/carfentanil, 4.2% screened positive for oxycodone/oxymorphone, and 11.2% screened positive for cocaine. Of the amphetamine class samples that screened positive, 95.3% confirmed positive and 4.7% confirmed negative. Of the benzodiazepine class samples that screened positive, 50.8% confirmed positive and 49.2% confirmed negative. Of the opiate class samples that screened positive, 79.8% confirmed positive and 20.2% confirmed negative. Of the fentanyl/carfentanil samples that screened positive, 97.2% confirmed positive and 2.8% confirmed negative. Of the oxycodone/oxymorphone samples that screened positive, 58.5% were confirmed positive and 41.5% confirmed negative. Of the cocaine samples that screened positive, 99.8% confirmed positive and 0.2% confirmed negative.

Conclusion
Our findings determined there were discrepancies among all classes when comparing immunoassay vs mass spectrometry results. There was good agreement between immunoassay and mass spectrometry results in the detection of fentanyl/carfentanil, cocaine and amphetamines. The differences between methods were most significant in the benzodiazepine, opiate and semi-synthetic opioid classes. This may be due to different cut-off levels between methods, specificity limitations of immunoassays and/or changing drug use patterns resulting in mass spectrometry multiple reaction monitoring false negative results.
B-330 Homogeneous Enzyme Immunoassay for Hydrocodone
Authors: Chung K , Pham M , Houts T , O’Malley R 
Published: September 2023

Abstract

Background
Hydrocodone is a semi-synthetic derivative of codeine and produces opioid-like effects similar to morphine. Hydrocodone (Trade names: Vicodin, Lortab, Hycodan, Vicoprofen) is one of the most frequently prescribed opioids in the U.S. as an antitussive (cough suppressant) and narcotic analgesic agent for the treatment of moderate to severe pain. Its ease of prescription and opioid-like effects has led to widespread drug diversion and abuse. The two commercially available homogeneous immunoassays for the detection of hydrocodone in urine at a cutoff of 300 ng/mL suffer undesirable cross-reactivity to oxycodone and morphine at concentrations below 25 000 ng/mL. ARK Diagnostics has developed the ARK Hydrocodone Assay to detect hydrocodone at a cutoff of 300 ng/mL with no cross-reactivity to oxycodone, morphine, and codeine at concentrations below 100 000 ng/mL.

Methods
The ARK™ Hydrocodone Assay is a liquid stable homogeneous enzyme immunoassay, consisting of two reagents, with a cutoff of 300 ng/mL and semi-quantitative range up to 800 ng/mL. The performance of this assay was evaluated on the Beckman Coulter AU680 Automated Clinical Chemistry Analyzer. Precision, spiked recovery, specificity, Histogram Overlap Analysis of ±25% controls and the cutoff, and method comparison with LC-MS/MS were evaluated.

Results
In semi-quantitative mode, total precision ranged from 3.9% to 9.9%CV. Spiked recovery of hydrocodone ranged from 86.2% (720 ng/mL) to 102.9% (240 ng/mL). The active metabolite, hydromorphone, showed an approximate equivalence to the 300 ng/mL hydrocodone cutoff at 299 ng/mL (100.3% cross-reactivity). Histogram overlap analysis showed no overlap between cutoff and control levels. Method correlation with LC-MS/MS using authentic urine samples showed an excellent agreement with a specificity of 100% and sensitivity of 96.9% (98 positives and 128 negatives).

Conclusion
The ARKTM Hydrocodone Assay measures hydrocodone and its metabolite hydromorphone in human urine with acceptable performance. The assay is sensitive, rapid, and applicable to a wide range of clinical chemistry analyzers.