bluuebunny/crossref_metadata_2025_split huggingface dataset:
B-221 Quantitation of DNA in Antibody-based and RNA-based Drugs by Quantitative Polymerase Chain Reaction (qPCR)
Authors: Su J , Uriu J , Aguilar E N , Tobias R , Marr G 
Published: September 2023

Abstract

Background
Current FDA regulation defined acceptance level of 10 ng per dose of residual DNA in drug products poses great challenges for accurate quantification of residual DNA, particularly for the products used in high doses or formulated in complex matrixes. The purpose of this study is to develop and qualify a qPCR method for the quantification of Chinese Hamster Ovary (CHO) DNA in antibody-based drugs and for quantitation of human DNA in RNA-based drugs.

Methods
An 8-point standard curve consisting of 0.005, 0.05, 0.5, 5, 50, 500, 1000 and 5000 pg CHO DNA and a 5-point standard curve consisting of 0.3, 3, 30, 300 and 3000 pg human DNA was generated by serial dilutions. The antibody sample (300 µg) was spiked with 2.5, 10, and 50 pg CHO DNA. The RNA sample (1.6 µg) was spiked with 0.3, 3 and 30 pg human DNA. The samples were analyzed by qPCR using a Real-Time PCR instrument. Commercial PCR test kits (TaqMan™ Probe and TaqMan™ Universal Master Mix), and sequence specific forward and reverse primers were used.

Results
For CHO DNA in antibody product, the linearity slope and R2 are −3.5 and 1.00, respectively, which met system suitability criteria. The accuracy by spiked recovery of 2.5 pg, 10 pg and 50 pg CHO DNA in 300 µg antibody, 150 pg CHO DNA in formulation buffer, and 150 pg CHO DNA in 1000 pg human DNA are 70%, 76%, 86%, 93% and 95%, respectively. The precision of the assay for 2.5 pg, 10 pg, and 50 pg of CHO DNA spiked in 300 µg antibody are 6%, 7% and 2%, respectively. The QL of the assay is 2.5 pg CHO DNA per 300 µg of antibody product.For human DNA in RNA product, the linearity slope and R2 are −3.3 and 1.00, respectively, which met system suitability criteria. The accuracy by spiked recovery of 0.3 pg, 3 pg, and 30 pg human DNA in 1.6 µg RNA, 3 pg human DNA in formulation buffer, 1.5 pg human DNA in 150 pg CHO DNA are 113%, 104%, 102%, 97% and 88%, respectively. The precision of the assay for 0.3 pg, 3 pg and 30 pg human DNA spiked in 1.6 µg RNA are 6%, 5% and 2%, respectively. The QL of the assay is 0.3 pg human DNA per 1.6 µg of RNA product.

Conclusion
Two qPCR methods were developed to quantify residual DNA in drug products. One method is specific to quantitation of low levels of CHO DNA in antibody product. Another method is specific to quantitation of low levels of human DNA in RNA product.
B-271 Molecular EQA Programs Verify the Use of a Quick Lysis Reagent in a Wide Range of Matrices and Pathogens
Authors: Martínez-Santolaria M , Sota-Diez C , Gaston-Galvez B , García-Manrique B , Machetti-Mareca E , Gil-Rodríguez C 
Published: September 2023

Abstract

Background
External Quality Assessment (EQA) programmes, like Quality Control for Molecular Diagnostics (QCMD), allow laboratories to verify the correct functionality of the molecular workflows used in the diagnosis of different pathogens. The aim of this study was to evaluate whether a quick molecular workflow had a good functionality for the diagnosis of different pathogens.

Methods
A total of 90 frozen samples from 2022 QCMD programmes were analysed. 20 transport medium (TM), 9 TM or saline and 1 saline samples from respiratory panels (QAV164188, QAV164189, QAM174193), 20 synthetic faecal matrix samples from gastrointestinal panels (QAP124154, QAB124153), 13 TM, 4 simulated swabs and 3 urines from sexual transmitted infections (STI) panels (QAB154177, QAM174201), and 18 TM and 2 synthetic cerebrospinal fluid from central nervous system (CNS) panels (QAV174195, QAM174196). Samples were processed using an in-house processing method called quick lysis reagent (QLR) (Certest Biotec S. L.). In parallel, samples were also extracted using the automatic robot KingFisher (Thermo Fisher Scientific). Nucleic acids were analysed at least in duplicate with qPCRs from VIASURE using the CFX96™ Real-Time PCR Detection System (Bio-Rad). Results were compared to the final diagnosis provided by QCMD and with the automatic extractor results.

Results
The in-house processing method QLR, in combination with VIASURE qPCRs, could process and identify bacteria, parasites and viruses from respiratory, gastrointestinal, STI and CNS panels. Negative samples were correctly detected. This workflow obtained a 100% of sensitive and specificity, same as the automatic extractor.

Conclusion
These EQA programmes tested the correct functionality of a quick molecular workflow, based on the VIASURE diagnostic products, that could detect different pathogens (bacteria, viruses, and parasites) from different origins (respiratory, gastrointestinal, STI and CNS).
B-299 Liquid Chromatography-mass Spectrometry Measurement of Drugs of Abuse and Alcohol Biomarker Phosphatidylethanol (peth 16:0/18:1) Using Volumetric Dried Blood Spot Device
Authors: Göksu Gürsu G , Maviş M E , Yılmaz H 
Published: September 2023

Abstract

Background
Reliable biomarkers have great significance in the investigation of alcohol consumption patterns and in the treatment of alcohol-induced diseases. Direct biomarkers to detect alcohol intake are non-oxidative products of ethanol metabolism (1, 2). Phosphatidylethanol (PEth), blood-based direct alcohol biomarker, represents a group of unnatural phospholipids formed on leukocytes and predominantly erythrocyte membranes. The presence of PEth in blood samples is an indisputable indicator of ethanol consumption (3, 4). Moreover, detecting drugs of abuse (DOA) including opiates, amphetamines, cocaine metabolites, benzodiazepines, THC-metabolites, MDMA, methadone, fentanyl etc. is important to establish patterns of illicit drug usage. In recent years, DBS has been gaining interest in plenty of research areas owing to ease of specimen collection and storage (5, 6).The main objective of this study was to determine the concentration of PEth-16:0 18:1 in DBS samples collected using a volumetric dried blood spot (DBS) device (HemaxisTM DB) and to identify/quantify drugs of abuse (including 119 parameters-Jasem Clinical Toxicology mixture) using another blood spot punch of the same DBS sample.

Methods
According to Jasem method, DBS samples were prepared by sampling from spiked 20 µL of blood drop located onto paraffin film. PEth-16:0 18:1 and DOA were extracted from entire blood spot of DBS specimens implementing two different extraction reagents at room temperature for 10 min then subjected to HPLC system equipped with Agilent 6470 TQ. The total run times from injection to injection for PEth and DOA were 4.0 min and 12.0 min respectively.

Results
The linearity and accuracy of the methods were evaluated using 5 DBS calibrators levels for PEth and 6 DBS calibrators levels for DOA. Linearity was confirmed in the range 10 to 1000 ng/mL (r² &gt; 0.995) for PEth 16:0/18:1 and in the range 2 to 100 ng/mL for DOA (r² &gt; 0.99). For two methods linearity, RSD% (inter-intraday) and accuracy were within the analytical acceptable ranges.

Conclusion
The measurement of blood PEth levels provides a precious tool to determine the chronological profile based on retrospective alcohol intake in a reliable way. Besides, simultaneous analysis of DOA containing 119 parameters ensures comprehensive analytical solution and clinically relevant information. Both of Jasem LC-MS/MS approaches are centred on simple sample preparation following non-invasive sample collection method. Prior to rapid extraction, applying volumetric DBS device assures to collect fixed volume of blood regardless of blood haematocrit level making the measurements more consistent.
B-307 Direct Quantitation of 77 Therapeutic and Clinical Toxicology Drugs in Dried Blood Spots Using the Fully Automated Transcend DSX-1 System
Authors: Guo J , Patterson C , Gibson R , van Natta K , Samra S 
Published: September 2023

Abstract

Background
The dried blood spot (DBS) sampling technique is advantageous over the traditional liquid blood collection due to its minimal invasiveness, smaller sample volume, improved analyte stability, and ease of storage and transportation, resulting in its increasing usage in therapeutic drug monitoring and clinical toxicology. Here, we describe a fully automated workflow to rapidly extract and quantify a wide range of therapeutic drugs and drugs of abuse in DBS using the Thermo Scientific™ Transcend™ DSX-1 system, fulfilling the routine drug monitoring needs in the clinical laboratories.

Methods
Calibrators in blood are spotted on the DBS cards, analytes are extracted with the innovative flow-through desorption technology, and their corresponding internal standards (IS) are introduced via an automated IS addition module in the dried spot autosampler. The 2-dimensional TurboFlow technology allows the removal of interferences from the extracted samples before analytical separation. An integrated software controls every step of the sample desorption and separation. Analyte quantitation is performed on the Thermo Scientific™ TSQ Altis™ Plus triple quadrupole mass spectrometer, and the data is analyzed in TraceFinder™ software.

Results
A total of 77 therapeutic and clinical toxicology drugs from eleven classes, including anticonvulsants, antidepressants, antihistamines, antipsychotics, benzodiazepines, cocaine, dissociatives, opioids, and stimulants, are quantified in a single injection from DBS cards using a rapid automated method on a Transcend DSX-1 system. DSX-1 combines a dried spot module for direct analyte extraction with Transcend™ UHPLC for online sample separation using the TurboFlow technology. The method only takes 4.3 min from analyte extraction to MS detection. Good calibration curves with R2 &gt; 0.98 are achieved using a weighting factor of 1/x, and the limit of quantification (LOQ) values are established with % RSD and % CV &lt; 15, │% Diff│ &lt; 20, and relative ion ratio &lt; %20. The LOQ values are all in the low ng/mL levels, which largely meet the screening and confirmation sensitivity needs of analytical methodologies in routine clinical laboratories.

Conclusion
A fully automated LC-MS-based workflow is developed to rapidly and reliably quantitate 77 compounds therapeutic and clinical toxicology drugs in dried blood spots.
B-316 Retrospective Data Analysis Reveals Unusual Metabolism Pattern of Ethanol in Pediatrics as Compared to Adult and Geriatric Populations
Authors: Baryeh K , Zhou R , Zuromski L M , McMillin G A , Krasowski M D , Johnson-Davis K L 
Published: September 2023

Abstract

Background
Ethanol is utilized in many pharmaceutical formulations and in the treatment of methanol poisoning. It is also consumed recreationally and is the most abused drug globally. About 95% of consumed ethanol is metabolized by alcohol dehydrogenase into acetaldehyde, followed by conversion of the acetaldehyde into acetic acid by acetaldehyde dehydrogenase. Less than one percent is metabolized via nonoxidative pathways, which may include: glucuronidation, sulfation, and the formation of fatty acid esters of ethanol. In neonates the glucuronidation pathway has been reported to be underdeveloped and matures with age. In this work, the patterns and concentrations of phase II nonoxidative metabolites of ethanol, ethyl glucuronide (EtG) and ethyl sulfate (EtS), were assessed in random urine collections of pediatric, adult, and geriatric patients.

Methods
Test results (n = 63498) from urine samples tested for EtG and EtS by quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) at our facility were utilized for this study. For both analytes, the lower limit of positivity was 100 ng/mL. The age range under consideration was 0–100 years. Data was partitioned into age groups: 0–17 years, 18–80 years and 81–100 years. The EtG and EtS concentrations across the partitions were compared.

Results
Across all ages, 60%–65% of patients had both EtG and EtS present in urine. Between 5%–10% had EtG present without EtS, and 25%–35% of patients had neither of the ethanol metabolites present. In all age groups there was a low positivity rate of EtS without the presence of EtG (0%–1%). All age partitions showed a similar pattern with the highest percentages of patients showing the presence of both metabolites, and the lowest percentage of patients showing the presence of EtS only. Markedly, there were no pediatric patient samples that had the sulfated metabolite present in the absence of the glucuronidated metabolite; this was statistically significantly different than this proportion in adults (Fisher’s Exact test, P = 0.025). This finding suggests greater glucuronidation activity in the pediatric population.

Conclusion
From the data presented in this work, EtG is more prevalent relative to EtS in urine samples of patients assessed for ethanol exposure. This work also shows that glucuronidation may be the predominant pathway for ethanol metabolism in pediatric populations. Although the level and frequency of ethanol exposure, as well as the time between exposure and sampling were unknown for the patients studied, the data offers insights into the relative distribution of EtG and EtS in random urine samples for pediatric and adult populations.
B-321 Development of Chemiluminescent Enzyme Immunoassay (CLEIA) for Detecting Methotrexate in Blood
Authors: Inagaki E , Baba K , Imai Y , Kaneko A , Aoyagi K 
Published: September 2023

Abstract

Background
Methotrexate (MTX) is a folate metabolism antagonist used in the treatment of rheumatoid arthritis, psoriasis, and antineoplastic agents. Therapeutic drug monitoring (TDM) is required to avoid serious adverse reactions of high-dose MTX/leucovorin rescue therapy or in patients with possible delayed excretion due to renal dysfunction or other factors. Although there are many reagents for MTX measurement based on the immunoassay method, they have a narrow measurement range to 2 µmol/L. Therefore, high concentration samples often make users conduct dilution and retest. In addition, most reagents have a cross-reactivity rate of over 40% with 2,4-diamino-N10-methylpteroic acid (DAMPA), one of the metabolites of MTX, so users must take note in the interpretation of measurement results. We have established an antibody with high specificity against MTX, and have developed a fully-automated, chemiluminescence immunoassay for LUMIPULSE® L2400 system for detecting MTX in blood, Lumipulse Presto® Methotrexate, which can significantly improve the issues described above.

Methods
MTX assay is a one-step competitive immunoassay on the LUMIPULSE® L2400 System (FUJIREBIO INC.), which requires 10 µL of sample. The resulting reaction signals are derived within 30 min/sample, and are proportional to the amount of MTX in the sample allowing quantitative determination of MTX in plasma or serum.

Results
The detection limit of the assay was 0.019 µmol/L, and the limit of quantitation was 0.030 µmol/L. A 20-day precision study was performed during a 31-day period using two controls and three panel specimens, and within-laboratory precision showed CV ≤ 3.1 %. Linearity was demonstrated over the range 0.017–28.821 µmol/L. No interference was observed with unconjugated (≤20.1 mg/dL) or conjugated bilirubin (≤20.2 mg/dL), chyle (≤1770 FTU), hemoglobin (≤450 mg/dL), and RF (rheumatoid factor, ≤5522 IU/mL). The correlation coefficient and the regression slope of this assay and LC-MS/MS were 1.00 and 1.0, respectively (N = 171). Compared to existing immunoassay reagents, the assay showed much lower cross-reactivity to DAMPA.

Conclusion
The performance of our novel MTX assay using LUMIPULSE® L2400 system was satisfactory, suggesting it is useful for routine analysis. In particular, the measurement range is wide, 0.030 to 25.000 µmol/L, which greatly reduces the frequency of dilution and retest of high concentration samples. Used with the auto dilution system in LUMIPULSE® L2400, it is possible to measure almost all clinical specimen automatically. Because of the low cross-reactivity with DAMPA, it is expected to be utilized in patient receiving glucarpidase.
Acknowledgment: Some of the samples were provided in accordance with “Guidelines for clinical measurement and diagnosis technology improvement project”. The guidelines are promoted by Tsukuba Medical Laboratory of Education and Research Center, and University of Tsukuba Hospital, Japan.
B-322 Assessment of MassTrak™ Immunosuppressant Calibrator and Control Sets: Confidence in Your Calibrators
Authors: Hammond G , Balloch S , Calton L 
Published: September 2023

Abstract

Background
The analytical performance of the Waters MassTrak Immunosuppressant Calibrator and Quality Control Sets (IVD) were evaluated using a laboratory developed LC-MS/MS method, to analyze all four immunosuppressants in a single run.

Methods
The MassTrak Immunosuppressant Calibrator and Quality Control Sets were used to quantify cyclosporine, everolimus, sirolimus and tacrolimus in human whole blood. Samples (50 µL) were treated with zinc sulfate and acetonitrile containing internal standards. A water/methanol/ammonium acetate gradient was used with a Waters C18 HSS SB Column on a Waters ACQUITYTM UPLTM I-Class FL and Xevo TQ-S micro Mass Spectrometer with an injection-to-injection time of less than 2 min.

Results
No system carryover was observed from high concentration samples. Precise quantification (≤20% CV, ≤15% bias) at concentrations equal to or lower than the lowest concentration calibrator was demonstrated. Total precision and repeatability (3 pools, 5 replicates, 5 days; n = 25) were determined to be ≤7.1% CV. The method was linear over the measuring interval of 19.3–1500 ng/mL (cyclosporine) and 0.77–39 ng/mL (everolimus, sirolimus and tacrolimus). Addition of high concentrations of several endogenous materials did not affect quantification. External quality assurance samples for all drugs met the scheme acceptance criteria (cyclosporine: n = 39, range 0–2658 ng/mL, mean % bias +1.0%; everolimus: n = 35, range 0–21.9 ng/mL, mean % bias +0.9%; sirolimus: n = 34, range 0–74.2 ng/mL, mean % bias −7.5%; tacrolimus: n = 40, range 0–23.0 ng/mL, mean % bias −0.5%).

Conclusion
Performance of the Waters MassTrak Immunosuppressant Calibrator and Quality Control Sets, with the developed method described provide good method performance. The accuracy data demonstrates confidence in the MassTrak Immunosuppressant Calibrator Set, providing harmonization across laboratories. This method is an example of an application using the instrumentation, software and consumables described in this document. This method has not been cleared by any regulatory entity for diagnostic purposes. The end user is responsible for completion of the method development and validation. MassTrak Immunosuppressant Calibrator and Quality Control Sets are not available for sale in all countries.
B-342 Fentanyl Urine Drug Screen Comparison of the ARK II and Lin Zhi Immunoassays
Authors: Lam K , Menlyadiev M , Suhandynata R , Fitzgerald R , Song L , Metushi I 
Published: September 2023

Abstract

Background
Given the opioid epidemic, screening for fentanyl in urine has become increasingly important. Routine opiate immunoassays cannot identify fentanyl, given its structural variance from the more common opiates such as morphine and codeine. Therefore, the detection of fentanyl by immunoassay urine drug screen depends on antibody specificity for fentanyl. The ARK II is a widely used immunoassay that will detect fentanyl and norfentanyl. Recently, a novel fentanyl assay by Lin-Zhi has become available on the Roche platform; here, we evaluate its performance.

Methods
One hundred twenty nine urine samples were analyzed for fentanyl across the Lin-Zhi and ARK II assay on the Cobas c502 platform. Samples were either analyzed immediately upon request for drug of abuse screening or frozen for subsequent analysis. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) and/or prescription was used as confirmation for the presence of fentanyl. Our of the total 129 tested samples, so far 59 have been confirmed and additional studies are ongoing. The mass spectrometry assay has a limit of detection of 1 ng/mL for fentanyl/norfentanyl. The ARK II immunoassay has a 1 ng/mL cut-off for fentanyl and 7% cross-reactivity for norfentanyl; the Lin Zhi has a 5 ng/mL cut-off for norfentanyl and 131.58% cross reactivity for fentanyl.

Results
There were 9/129 discrepant samples between ARK II and Lin Zhi immunoassays as shown in Fig. 1. From the 59 confirmed samples Ark II resulted in one false positive sample and seven false negative samples (Fig. 1B) while the Lin Zhi assay resulted in 9 false negatives.

Conclusion
Given the current data set, there appear to be some discrepant results between the ARK II and Lin Zhi immunoassays. Most of the discrepant results observed have concentrations of fentanyl and norfentanyl that are close to the lower limit of immunoassay detection.
B-375 Mer-idpcr: A Method for Sequence-specific Measurement of Small RNA Expression and Methylation in Liquid Biopsies
Authors: Horos R , Daniel-Moreno A , Bieg-Salazar C , Skottke J , Ceiler J , Sanchez-Delgado M , Sikosek T , Rajakumar T , Steinkraus B R 
Published: September 2023

Abstract

Background
Small RNAs are an important class of biomarkers in liquid biopsy for cancer diagnostics. New discoveries in the field of “epitranscriptomics” have uncovered further functional layers of tumor biology that are ignored by standard gene expression measurement methods. Therefore, there is a pressing need for sequence specific methods that can detect methylation stoichiometry on small RNAs purified from liquid biopsies in a diagnostic setting.

Methods
To detect small RNAs specifically, we designed structured oligonucleotide adapters that bear reverse complementary overhangs to both the 5′ and 3′ end of target RNAs. After nick-based ligation, reverse transcription under limiting dNTP conditions enables the differential processing of methylated vs unmethylated species. Finally, target specific TaqMan probes are used on a digital PCR based platform for final quantification and calculation of methylation stoichiometry.

Results
We show that mer-idPCR can detect target RNAs in a sequence-specific manner using synthetic cognate RNA oligos and specificity controls, such as nucleotide variants or non-target miRNAs. Detected RNA is quantified in a linear range with a limit of detection at 40 copies/µL of cDNA. Concurrently, the method can detect 2′-o-methylation in 0%–100% range with a resolution of 10%. mer-idPCR can be applied to RNA extracted from various sources including serum, plasma or whole blood stabilizing tubes.

Conclusion
mer-idPCR can enable the high resolution, sequence specific quantification of target small RNA species, in addition to the measurement of 2′-o-methylation site status, in small RNAs extracted from peripheral blood. We show the adapter design requirements and discuss optimizations of adapter design and reverse transcription conditions that led to improved specificity, reproducibility, and linearity of this method. mer-idPCR enables the validation and discovery of novel biomarkers for cancer detection in liquid biopsies.